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            July Week 2  :  09.09.19  to
            09.13.19

        
        09.09.19
        Bacteria culture - E. coli containing the plasmid
          PET28:GFP 
        In order to extract the plasmid PET28:GFP, E. coli
          was cultured on LB petri dishes containing kanamycin at 50 µg/mL. The
          cultures were stored all night at 37°C. 
        Bacteria culture - E. faecium and S. aureus
        E. faecium and S. aureus were grown until
          the OD reached between 0.5 and 0.6.
         Digestion - EFr2 & SAr2
        PCR samples from the Round 2 of SELEX were digested using
          lambda exonuclease. A mix was performed in order to scale-up this step
          and get enough digested material. 

        
        1. Prepare the mix as
          follows
        
          
            
              

              
              50µL reaction
              Mix
            
            
              DNA (PCR11)
              4 µg
              120 µg
            
            
              Lambda exonuclease buffer (10X)
              5µL
              µL
            
            
              Lambda exonuclease
              1µL
              30µL
            
            
              Nuclease-free H²0
              QSP 50 µL
              QSP 1.5 mL
            
          
        
        

        2. Incubate samples for 30
          min at 37°C in a water-bath
        3. Stop the reaction by
          adding EDTA to 10 mM
        4. Heat at 75°C for 10
          minutes to inactivate the enzyme
        Samples concentrations were checked using a Nanodrop 2000
          spectrophotometer.
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              Dig-Round 2 - SA
              32.6
              1.91
              0.27
            
            
              Dig - Round 2- EF
              20
              1.92
              0.25
            
          
        
        

        Phenol/chloroform extraction with Isopropanol - Digested
          products EFr2 & SAr2 
        Once digested, samples were purified after digestion.
          They were analysed with a Nanodrop 2000 spectrophotometer.
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              EFr2 sample 1
              46.7
              1.47
              1.49
            
            
              EFr2 sample 2
              42.1
              1.48
              1.52
            
            
              SAr2 sample 1
              29.1
              1.66
              1.63
            
            
              SAr2 sample 2
              37.3
              1.52
              1.55
            
          
        
        

        Interpretation
        A total quantity of 692 pmol was obtained for E.
            faecium in the Round 2, and 516 pmol for S. aureus
        SELEX Round 3
        The first round of SELEX was performed with 1h45 hours of
          incubation. The samples are then analyzed using a Nanodrop 2000
          spectrophotometer.
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              SAr3
              33.6
              2.19
              1.45
            
            
              EFr3
              42.9
              2.3
              1.76
            
          
        
        PCR 11 - Amplification of EFr3 and SAr3
         This PCR was performed to amplified the DNA after the
          Round 3 of SELEX. The PCR followed the same program as described
          previously with the same concentration. It was run overnight and hold
          at 4°C. The samples are analyzed using a Nanodrop 2000
          spectrophotometer. 
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              PCR 11 - Round 3 - SA
              1086.6
              1.67
              1.20
            
            
              PCR 11 - Round 3 - EF
              1496
              1.72
              1.38
            
          
        
        

        09.10.19
        Bacteria culture - E. coli containing the plasmid
          PET28:GFP
         After an overnight growth, a colony of bacteria from the
          09.09 was added into an erlenmeyer containing LB media. Then, the
          erlenmeyer was put at 37°C all night under agitation at 140 rpm. 
        Bacteria culture - E. faecium and S. aureus
        E. faecium and S. aureus were grown until
          the OD reached between 0.5 and 0.6 
        Digestion - EFr3 & SAr3
         PCR samples from the Round 3 of SELEX were digested
          using lambda exonuclease. A mix was performed in order to scale-up
          this step and get enough digested material. Samples concentrations
          were checked using a Nanodrop 2000 spectrophotometer. 
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              Dig - Round 3 - SA
              36.8
              1.92
              0.27
            
            
              Dig - Round 3 - EF
              45.3
              1.81
              0.23
            
          
        
        

         Phenol/chloroform extraction with Isopropanol and
          Glycogen - Digested products EFr3 & SAr3 
         Once digested, samples were purified after digestion
          using a phenol/chloroform extraction. Glycogen for a final 0.5 μg/μL
          concentration was added in order to maximise the amount of DNA
          recovered and avoid pipetting the pellet. They were analysed with a
          Nanodrop 2000 spectrophotometer. 
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              EFr3 sample 1
              32.1
              1.56
              1.86
            
            
              EFr3 sample 2
              22.7
              1.48
              1.89
            
            
              SAr3 sample 1
              27.1
              1.52
              1
            
            
              SAr3 sample 2
              65.4
              1.50
              1.24
            
          
        
        Interpretation
         A total quantity of 856 pmol was obtained for E.
            faecium in the Round 3, and 516 pmol for S. aureus.
          Because the purity of SAr3 sample 1 was really low, we only used SAr3
          sample 2 for the following steps. 
         09.11.19 
        Midiprep - E. coli containing the plasmid
          PET28:GFP 
         The liquid culture of E. coli from the 10.09 was
          centrifuged 10 minutes at 3,500 x g and the supernatant was removed.
          Then, we used a midiprep kit from Qiagen to extract the plasmid from E.
            coli. For that, the pellet was resuspended with 4 mL of P1
          containing RNAse. Then, 4 mL of P2 was added and we waited 5 minutes
          at RT. After that, 4 mL of P3 was added and the solution was mixed.
          The solution was loaded on a column and the filtrate was recovered.
          After, 4 mL of QBT Qiagen lip was added on the column and then the
          filtrate was loaded on the column. After filtration, the column was
          washed twice with QC buffer. In the filtrate, 5 mL of QF buffer and
          3.5 mL of isopropanol were added. The solution was centrifuged during
          30 minutes at 15000 x g. The supernatant was removed and the pellet
          was washed with 1 mL of ethanol 70% and centrifuged again 30 minutes
          at 15,000 x g. We removed all the supernatant and let the pellet dry.
          Then, 10 µL of TE buffer was added and the sample was vortexed.
          Finally, the sample was stored at -20°C. The sample was checked with a
          Nanodrop 2000 spectrophotometer. 
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              pET28:GFP
              212.6
              1.88
              2.24
            
          
        
        

        09.12.19 
         Bacteria culture - E. faecium and S. aureus
        
        E. faecium and S. aureus were grown until
          the OD reached between 0.5 and 0.6
        SELEX Round 4
         The Round 4 of SELEX was performed by incubating for
          1h30. The samples are then analyzed using a Nanodrop 2000
          spectrophotometer. 
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              SAr4
              37.1
              2.02
              1.40
            
            
              EFr4
              21.2
              2.25
              1.55
            
          
        
        

         PCR 12 - Amplification of EFr4 and SAr4 
         This PCR was performed to amplified the DNA after the
          Round 4 of SELEX. The PCR followed the same program as described
          previously with the same concentration. It was run overnight and hold
          at 4°C. The samples are analyzed using a Nanodrop 2000
          spectrophotometer. 
        

        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              PCR 12 - Round 4 - SA
              1373.9
              1.62
              1.05
            
            
              PCR 12 - Round 4 - EF
              1314.2
              1.62
              1.06
            
          
        
        

         09.13.19 
        Bacteria culture - E. faecium and S. aureus
         E. faecium and S. aureus were grown
          until the OD reached between 0.5 and 0.6 
         Digestion - EFr4 & SAr4 
         PCR samples from the Round 4 of SELEX were digested
          using lambda exonuclease. A mix was performed in order to scale-up
          this step and get enough digested material. Samples concentrations
          were checked using a Nanodrop 2000 spectrophotometer. 
        

        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              Dig - Round 4 - SA sample 1
              59.7
              1.79
              0.27
            
            
              Dig - Round 4 - SA sample 2
              59.2
              1.80
              0.25
            
            
              Dig - Round 4 - EF sample 1
              57.4
              1.80
              0.24
            
            
              Dig - Round 4 - EF sample 2
              58.8
              1.79
              0.24
            
          
        
        

         Phenol/chloroform extraction with Isopropanol and
          Glycogen - Digested products EFr4 & SAr4 
         Once digested, samples were purified after digestion.
          They were analysed with a Nanodrop 2000 spectrophotometer. 
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              EFr4 sample 1
              49.6
              1.46
              1.17
            
            
              EFr4 sample 2
              34.1
              1.51
              1.13
            
            
              SAr4 sample 1
              39.6
              1.54
              1.15
            
            
              SAr4 sample 2
              18.7
              1.54
              1.33
            
          
        
        

         Interpretation 
         A total quantity of 650 pmol was obtained for E.
            faecium in the Round 4, and 684 pmol for S. aureus.
         SELEX Round 5 
         The Round 5 of SELEX was performed by incubating for
          1h15. The samples are then analyzed using a Nanodrop 2000
          spectrophotometer. 
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              SAr5
              30.3
              1.70
              1.03
            
            
              EFr5
              33.7
              2.21
              1.74
            
          
        
        

         PCR 13 - Amplification of EFr5 and SAr5 
         This PCR was performed to amplified the DNA after the
          Round 5 of SELEX. The PCR followed the same program as described
          previously with the same concentration. It was run overnight and hold
          at 4°C. The samples are analyzed using a Nanodrop 2000
          spectrophotometer. 
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              PCR 13 - Round 5 - SA
              1228.2
              1.59
              0.99
            
            
              PCR 13 - Round 5 - EF
              1334.8
              1.62
              1.02
            
          
        
        

        
      
      
        
          
          
          
        
      
      
        
          
          
          
          
        
        
        	 
        
      
      
        
          
          
          
          
        
        
              July Week 4   :   07/22   to   07/28 
        




        07.22.19
        Resuspension of oligonucleotides  : 
        DNA library and primers were produced by Eurofins Genomics. Once received, they were resuspended according to the manufacturer’s instructions. Stock solutions of 100 µM were made by diluting the lyophilized oligonucleotides with a 10 mM Tris- 0.1 mM EDTA pH 8.0 solution (TE8.1). Concentrations were checked using a Nanodrop 2000 spectrophotometer. 


        
          
            
              Samples
              Concentration (ng/µl)
              260/280nm ratio
              260/230nm ratio
            
            
              DNA library
              2217.65
              1.89
              1.56
            
            
              Reverse primer
              8810.90
              1.95
              1.58
            
          
        
        

         PCR 1  -  Amplification of the DNA library 
         In order to start the SELEX, we need to amplify our DNA library in order to get enough material to start with. We tried several PCR protocols to optimize the amplification. This test was performed in order to know if the magnesium concentration could impact the quality of the amplification, and different annealing temperatures from 44 to 54°C were tested. A mix of Taq DNA polymerase and phusion DNA polymerase was used. 
        
          
            
            
            Volume needed (μL)
            
            
          
          
          	Components
            Final concentration
            Mix 1
            Mix 2
            Mix 3
          
          
            Phusion HF buffer 5X
            1X
            50
            50
            50
          
          
            25 mM dNTPs
            0.20  mM
            2
            2
            2
          
          
            Forward primer
            0.50  μM
            9.75
            9.75
            9.75
          
          
            Reverse primer
            0.50  μM
            10.10
            10.10
            10.10
          
          
            25 mM Magnesium
            2.5  mM
            25.0
            50.0
            75.0
          
           
            DNA library
            10.0  ng
            22.5
            22.5
            22.5
          
          
            Taq DNA polymerase
            0.10  U/μL
            0.02
            0.02
            0.02
          
          
            Phusion DNA polymerase
            1    U/μL
            0.50
            0.50
            0.50
          
          
            Nuclease-free water
            /
            130.13
            105.13
            80.13
          
          
            Total volume
            /
            250
            250
            250
          
        
        

        
          Rq: Enzymes were added at the end as hot-start PCR protocols can optimize the yield of the desired product while limiting the likelihood of nonspecific amplification
        
        

        
          PCR plate plan
        
        
          
            A (54°C)
            1.1
            2.1
            3.1
          
          
            B
            /
            /
            /
          
          
            C (51.9°C)
            1.2
            2.2
            3.2
          
          
            D
            /
            /
            /
          
          
            E (47.6°C)
            1.3
            2.3
            3.3
          
          
            F
            /
            /
            /
          
          
            G (44.7°C)
            1.4
            2.4
            3.4
          
          
            G
            /
            /
            /
          
        
        


        
          PCR program used:
        

        
          -    95°C             5 min

          
          -    95°C             30 s
        
          -    44-54°C      1 min        30 cycles
        
          -    68°C             30 s      
        
          -    68°C             5 min
          
          -    15°C             HOLD ALL NIGHT
        
        

        
          PCR was run overnight. Samples were checked using a Nanodrop 2000 spectrophotometer.
        
        

        
          
            Sample
            Concentration (ng/μL)
            260/280nm ratio
            260/230nm ratio
          
          
          	1.1
            465.90
            1.39
            0.66
          
           
          	1.2
            501.7
            1.42
            0.69
          
          
          	1.3
            480.4
            1.43
            0.68
          
          
          	1.4
            469.9
            1.43
            0.65
          
          
          	2.10
            498.30
            1.40
            0.68
          
          
          	2.20
            498.70
            1.43
            0.70
          
          
          	2.30
            532.90
            1.42
            0.71
          
          
          	2.40
            495.20
            1.42
            0.69
          
          
          	3.10
            497.80
            1.43
            0.70
          
          
          	3.20
            512.20
            1.43
            0.70
          
          
          	3.30
            505.50
            1.42
            0.70
          
          
          	3.40
            527.20
            1.41
            0.70
          
        
        

        
          Interpretations :
        
        
          No special condition seemed better than others.
        
        




        
          07.23.19
        
        
          Gel electrophoresis 1  -  Amplified library
        


        
          To verify that the samples were all the same with the right size, we performed a gel electrophoresis using a 3% agarose gel. Each band was cut and then kept at -4°C in order to be purified later.
        
        

        
          
            
          
          
            
              
                1.
                Sample 1.1 (54°C, 25  μL, Mg2+)
              
              
                2.
                   Sample 1.2 (51.9°C, 25  μL, Mg2+)
              
              
                3.
                   Sample 1.3 (47.6°C, 25  μL, Mg2+)
              
              
                4.
                   Sample 1.4 (44.7°C, 25  μL, Mg2+)
              
               
                5.
                Sample 2.1 (54°C, 50  μL, Mg2+)
              
              
                6.
                   Sample 2.2 (51.9°C, 50  μL, Mg2+)
              
              
                7.
                   Sample 2.3 (47.6°C, 50  μL, Mg2+)
              
              
                8.
                   Sample 2.4 (44.7°C, 50  μL, Mg2+)
              
              
                9.
                Sample 3.1 (54°C, 75  μL, Mg2+)
              
              
                10.
                   Sample 3.2 (51.9°C, 75  μL, Mg2+)
              
              
                11.
                   Sample 3.3 (47.6°C, 75  μL, Mg2+)
              
              
                12.
                   Sample 3.4 (44.7°C, 75  μL, Mg2+)
              
            
          
          


         
        
         
            Interpretations
          
          
            All samples had the same size of 120 bp approximately whereas the library size was 80 bp normally. 
          
          


          
            Conclusion
          
          
            PCR amplification resulted in a shift in size of the oligonucleotides because of the double-stranded form.
          
        

        

        
          Cell culture
        
        
          TO COMPLETE
        
        
          07.24.19
        
        
          Growth curves
        
        

        
          Bacteria were cultured and their growth was analyzed by establishing a growth curve associated with the number of colonies. Several strains were used:
        
        
          
E. faecium (ref CIP 103014T)
          P. aeruginosa (ref 104116)
          A. baumanii (ref CIP 79.34T)
          S. aureus (ref CIP 103429)
          S. pneumoniae (ref CIP 104340)
          E. coli (ref 53.126)
          E. enterica (ref CIP 60.62T)
          


        
        
          All strains were provided by the CRBIP of the Institut Pasteur.
        
        
          TO COMPLETE
        
        
          07.25.19
        
        
          Purification of PCR products  -  Invitrogen Pure Link Quick Gel extraction kit  -  PCR 1
        
        
          After PCR amplification, PCR products need to be cleaned up. Gel slices were extracted and purified using Invitrogen Pure Link Quick Gel extraction kit following the manufacturer’s instructions. 
        
        
          
        
        




        
          END OF THE WEEK
        
      
      
      
        
          
          
          
          
        
        
            July Week 5   :   07/29
            to   08/04 
        




         07.29.19
         Gel electrophoresis 2  -  Purified PCR products from gel extraction kit 


        To verify that the samples were purified after gel extraction, we performed a gel electrophoresis using a 3% agarose gel. We used two ladders, one at 25 bp and the other at 50 bp from Promega. 
        


        
          
        
        


        
          Interpretation
        
        
          There was no band after purification by gel extraction. So, during the purification step we lost the DNA.
        
        


        
          Digestion  -  Amplified library
        
        
          This step is made to pass from a double-stranded DNA to a single-stranded one. 
We performed the digestion using Lambda Exonuclease from New England Biolabs directly on the amplified library (PCR 1) without purifying it beforehand.
        
        
          1.   Set-up the reaction as follows:
        
        
        
          
            Components
            Volume for 50  μL reaction
          
          
            DNA from PCR 1
            5  μg
          
          
            Lambda Exonuclease Reaction Buffer (10X)
            5 μL  (1X)
          
          
            Lambda Exonuclease
            1 μL  (5 units)
          
          
            Nuclease-free water
            up to 50μL
          
        
        
          2.   Incubate at 37°C for 30 minutes.
        
        
          3.   Stop reaction by adding EDTA to 10 mM.
        
         
          4.   Heat at 75°C for 10 minutes.
        



        
          07.31.19
        
        
          PCR 2  -  Testing different conditions
        
        

        
          To try to enhance our PCR protocol, we performed a new PCR using different conditions. These conditions enabled us to determine if the Taq DNA Polymerase has to be activated prior addition, and if both polymerases have to be added at 95°C or not. The PCR was run overnight and hold at 4°C.
        
        
          
            
            Volume needed (in μL)
            
          
          
            Components
						Mix 1
            Mix 2
          
          
            Phusion HF buffer 5X
						500
            500
          
          
            25 mM dNTPs
						20
            250
          
          
            Forward primer
						97.5
            50
          
          
            Reverse primer
						101
            50
          
          
            25 mM Magnesium
						250
            250
          
          
            DNA library (1/1000 dilution)
						225
            250
          
          
            Taq DNA polymerase diluted 1/10
						5
            5
          
          
            Phusion DNA polymerase
						25
            25
          
          
            Nuclease-free water
						1276.5
            1135
          
          
            Total volume
						2500
            2500
          
        
        

        
          PCR plan
        
        
          
            
            1
            2
            3
            4
            5
            6
            7
            8
            9
            10
            11
            12
          
          
            A
            1A
            2A
            3A
            4A
            5A
            6A
            7A
            8A
            9A
            10A
            11A
            12A
          
          
            B
            1B
            2B
            3B
            4B
            5B
            6B
            7B
            8B
            9B
            10B
            11B
            12B
          
          
            C
            1C
            2C
            3C
            4C
            5C
            6C
            7C
            8C
            9C
            10C
            11C
            12C
          
          
            D
            1D
            2D
            3D
            4D
            5D
            6D
            7D
            8D
            9D
            10D
            11D
            12D
          
          
            E
            1E
            2E
            3E
            4E
            5E
            6E
            7E
            8E
            9E
            10E
            11E
            12E
          
          
            F
            1F
            2F
            3F
            4F
            5F
            6F
            7F
            8F
            9F
            10F
            11F
            12F
          
          
            G
            1G
            2G
            3G
            4G
            5G
            6G
            7G
            8G
            9G
            10G
            11G
            12G
          
          
            H
            1H
            2H
            3H
            4H
            5H
            6H
            7H
            8H
            9H
            10H
            11H
            12H
          
          
            
            Mix 1
            Mix 2
          
        
        


        
          Legend  :
        
        
          
Condition 1: Taq was activated and added with Phusion in ice
          Condition 2: Taq was not activated and added with Phusion in ice
          Condition 3: Phusion was added in ice
          Condition 4: Taq was activated and added with Phusion at 95°C
          Condition 5: Taq was not activated and added with Phusion at 95°C
          Condition 6: Phusion was added at 95°C
          Condition 7: Taq was activated and added with Phusion in ice
          Condition 8: Taq was not activated and added with Phusion in ice
          Condition 9: Phusion was added in ice
          Condition 10: Taq was activated and added with Phusion at 95°C
          Condition 11: Taq was not activated and added with Phusion at 95°C
          Condition 12: Phusion was added at 95°C
        
        

        
          Gel electrophoresis 4  -  PCR 2
        
        
          A gel containing 3% of agarose was run to determine which condition led to the best results.
        
        
          
        
        

        

        
          Interpretation
        
        

        
          Condition 1 provided the best results because the band is the brightest. Moreover, there was almost no difference between the samples containing activated Taq polymerase and when the polymerases were added on ice or in 95°C. Because we obtained the same results when we amplified using both Taq and phusion polymerases and only Phusion polymerase, we decided to only use Phusion polymerase for our PCR.
        
        


        
          08.01.19
        
        
          DNA precipitation  -  Product of phenol/chloroform from 29.07.19
        
        
          1.   Add 1/10 volume of Sodium Acetate (3 M, pH 5.2) and 2,5x volume of 100% ethanol
        
        
          
            Sample
            Pool 1 Elution 1
            Pool 1 Elution 2
            Pool 2 Elution 1
            Pool 2 Elution 2
          
          
            Upper aqueous phase recovered
            2000 µL
            430 µL
            1150 µL
            415 µL
          
           
            Sodium acetate 3M to add
            200 µL
            43 µL
            115 µL
            41.5 µL
          
          
            Volume after addition of sodium acetate
            2200 µL
            473 µL
            1265 µL
            456.5 µL
          
          
            Ethanol 100%  to add
            5500 µL
            1182.5 µL
            3162.5 µL
            1141.25 µL
          
          
            Total volume
            7700 µL
            1655.5 µL
            4427.5 µL
            1597.75 µL
          
        
        

        
          2.   Place the tube at -20°C overnight to precipitate the DNA from the sample. The experiment was continued on 08.02.19
        
        


        
          08.2.19
        
        

        
          DNA precipitation
        
        
          1.    Centrifuge the sample at 4°C for 30 minutes at 16,000 × g to pellet the cDNA.
          2.    Carefully remove the supernatant without disturbing the cDNA pellet.
          3.    Add 150 μL of 70% ethanol.
          4.    Centrifuge the sample at 4°C for 15 minutes at 16,000 × g. Carefully remove the supernatant.
          5.    Dry the cDNA pellet at room temperature 
          6.    Resuspend the cDNA pellet in 300 μL of TEN buffer by pipetting up and down
        
        

        
          Purification of PCR product  -  illustra MicroSpin G-25 Columns kit from GE Healthcare - PCR 2 
        
        
          Because the purification by gel extraction was not conclusive, we tested a second method of purification using the illustra MicroSpin G-25 Columns kit from GE Healthcare. The experiment was performed according to the manufacturer’s recommendation.
        
        


        
          Gel electrophoresis 5  -  Purified products from gel extraction vs. columns
        
        
          Because the purification by gel extraction was not conclusive, we tested a second method of purification using the illustra MicroSpin G-25 Columns kit from GE Healthcare. The experiment was performed according to the manufacturer’s recommendation.
        
        
          
        
        


        
          Interpretation
        
        
          We only observed the bands from the samples purified with the columns. Moreover, they seemed well purified because there was almost no smear.
        
        

        
          PCR 3  -  Testing different conditions (with/without DMSO + new program)
        
        
          To try to enhance our PCR protocol, we performed a new PCR using two conditions, one with DMSO and the other without . The PCR was run overnight and hold at 4°C.
        
        

        
          
            
            Volume needed (in μL)
            
          
          
            Components
            Mix 1 (With DMSO)
            Mix 2 (Without DMSO)
          
          
            Phusion HF buffer 5X
            50
            50
          
          
            25 mM dNTPs
            5
            5
          
          
            Forward primer
            10
            10
          
          
            Reverse primer
            10
            10
          
          
            DMSO
            7.5
            0
          
          
            Template DNA
            22.5
            22.5
          
          
            Phusion DNA polymerase
            2.5
            2.5
          
           
            Nuclease-free water
            142.5
            150
          
          
            Total volume
            250
            250
                   
        
        
      
      
        
          
          
          
          
        
        
           
          August Week 1  :  08/05   to  08/11
        
        
          08.05.19
        
        

        
          Purification of PCR products  -  illustra MicroSpin G-25 Columns kit from GE Healthcare  -  PCR 3
        
        
          Once again, we tried to purify our PCR products using different methods in order to compare them. This experiment was performed according to the manufacturer’s protocol.
        
        

        
          Purification of PCR products  -  PCR clean-up kit from Quiagen  -  PCR 3
        
        
          This experiment was performed according to the manufacturer’s protocol.
        


        
          Gel electrophoresis 6  -  Purified products from gel extraction (30.07.19) vs. GE Healthcare vs. Quiagen
        
        
          To analyze the different purification methods of our PCR products from 02.08 and the effect of DMSO, we ran the samples on an 3% agarose gel. Moreover, we compared the percentage of agarose in gel between 3 and 4% to see if the band resolution was better.
        
        
          
        
        

        
          1.    PCR product from 02.08 with DMSO
          2.    PCR product from 02.08 without DMSO
          3.    PCR product with DMSO purified with Qiagen kit
          4.    PCR product whithout DMSO purified with Qiagen kit
          5.    PCR product with DMSO purified with GE Healthcare column
          6.    PCR product without DMSO purified with GE Healthcare column
          7.    PCR product with DMSO purified by gel extraction
          8.    PCR product without DMSO purified by gel exctraction
        
        

        
          
            Wells
            Purification method
            Concentration (ng/μL)
            260/280 ratio
            260/230 ratio
          
          
            4
            PCR product with DMSO
            1157.6
            1.64
            0.85
          
          
            5
            PCR product without DMSO
            1143
            1.65
            1.18
          
          
            6
            PCR clean up kit from QIAGEN with DMSO
            7.9
            2.26
            -0.32
          
          
            7
            PCR clean up kit from QIAGEN without DMSO
            15
            2.07
            -0.62
          
          
            8
            GE Healthcare columns with DMSO
            34.5
            0.96
            0.34
          
          
            9
            GE Healthcare columns without DMSO
            45.9
            1.16
            0.08
          
          
            10
            Gel extraction kit with DMSO
            6.1
            -6.66
            0.01
          
          
            11
            Gel extraction kit without DMSO
            -0.5
            0.53
            0
          
        
        

        
          Interpretations
        
        
          The gel containing 4% of agarose gave a better resolution of the bands. Moreover, we did not observe any difference between the amplified products with and without DMSO. Concerning the purification methods, there was still no bands after the purification by gel extraction. Concerning the purification by PCR clean-up, we did not see anything on the gel at 4% agarose and there was only one band in the well 9 on the gel with 3% of agarose. On the contrary, we observed a band on both gels  for the two products purified by the columns. 
        
        
          We analyzed then the same products as above but after digestion. The digestion was performed according to the manufacturer’s protocol.  This time we only did a 4% of agarose gel because we noticed with the previous experiment that we had a better resolution with this concentration.
        
        
          
        
        

        
          Interpretation
        
        
          After digestion, there was no more bands even for the sample purified with the columns. Thus, either the lambda exonuclease had digested the two strands of DNA, or the concentration of DNA was too low to be seen on the gel. The last hypothesis was that the SYBR Green may not reveal single-stranded DNA.
        
        

        
          Conclusion
        
        
          Several parameters may change during the digestion step in order to determine if the enzyme digested the two strands of DNA.
        
        

        
          Phenol/chloroform extraction
        
        
          We performed a purification step on all samples to remove the digestion enzyme according to the protocol previously described. Samples were then analyzed using a Nanodrop 2000 spectrophotometer.
        
        

        
          
            Wells
            Purification method
            Concentration (ng/μL)
            260/280 ratio
            260/230 ratio
          
          
            5
            PCR product with DMSO
            1157.6
            1.64
            0.85
          
          
            6
            PCR product without DMSO
            1143
            1.65
            1.18
          
          
            8
            PCR product clean up kit from QIAGEN with DMSO
            7.9
            2.26
            -0.32
          
          
            9
            PCR product clean up kit from QIAGEN without DMSO
            15
            2.07
            -0.62
          
          
            10
            GE Healthcare columns with DMSO
            34.5
            0.96
            0.34
          
          
            11
            GE Healthcare columns without DMSO
            45.9
            1.16
            0.08
          
          
            13
            Gel extraction kit with DMSO
            6.1
            -6.66
            0.01
          
          
            14
            Gel extraction kit without DMSO
            -0.5
            0.53
            0
          
        
        

        
          07.08.19
        
        

        
          PCR 4  -  15 cycles vs 30 cycles
        
        
          We tried to amplify our library with only 15 cycles instead of 30 to compare if it had an impact in the shift of size of our PCR product (120 bp instead of 80 bp normally). The PCR was made with the same conditions as the PCR 4 without DMSO. 
        
        
          Gel electrophoresis 7  -  15 cycles vs 30 cycles
        
        
          The amplified products were run on a 4% agarose gel and compared with amplified products from PCR 3 and 4.
        
        

        
          
        
        
          Interpretations
        
        
          We observed a slight decrease of the size for products amplified with only 15 cycles compared to those amplified with 30 cycles. However, this decrease in size was not significant enough. And the bands from the amplified samples with 30 cycles were brighter than those amplified with 15 cycles.
        
        

        
          Purification of PCR products  -  Dialysis tubing
        
        
          A new purification method of our PCR products was tested which is dialysis tubing. The DNA will migrate out of the gel and into the dialysis bag. Samples were checked using a Nanodrop 2000 spectrophotometer. 
        
        

        
          
          	Sample
            Concentration (ng/μL)
            260/280 ratio
            260/230 ratio
          
           
          	Dialysis sample 1 pool
            1.2
            2.71
            -9.20
          
           
          	Dialysis sample 2 pool
            2.8
            2.22
            0.6
          
        
        

        
          Interpretation
        
        
          Even if the ratio 260/280 was good the concentration of DNA was too low. Thus, this technique cannot be used due to a yield too low.
        
        

        
          Kit montage Gel Extraction
        
        
          We used the Kit montage Gel extraction from Millipore to try to extract our DNA from the gel and purify it. This kit was used according to the manufacturer’s protocol. Samples were checked using a Nanodrop 2000 spectrophotometer. 
        
        

        
          
            Sample
            Concentration (ng/μL)
            260/280 ratio
            260/230 ratio
          
          
            Sample 1
            8.3
            1.95
            0.15
          
          
            Sample 2
            6.4
            2.13
            0.16
          
          
            Sample 3
            5
            2.24
            0.12
          
          
            Sample 4
            6.9
            1.37
            0.14
          
        
        

        
          Interpretation
        
        
          The concentration of DNA was too low. Hence, another method of DNA purification has to be found.
        
        
      
      
        
          
          
          
          
        
        
                    August Week 2 :  07/12  to  07/18 
         07.12.19 
         Digestion  -  Testing different conditions on
          amplified library 
         A new digestion was carried out in order to optimize the
          yield. Two parameters were studied, the incubation time and the
          quantity of enzyme used. Here is the list of all conditions tested: 
        1A : 5 min incubation, 5 U
        1B : 5 min incubation, 2.5 U
        1C : 5 min incubation, 1 U
        2A : 10 min incubation, 5 U
        2B : 10 min incubation, 2.5 U
        2C : 10 min incubation, 1 U
        3A : 15 min incubation, 5 U
        3B : 15 min incubation, 2.5 U
        3C : 15 min incubation, 1 U
        4A : 20 min incubation, 5 U
        4B : 20 min incubation, 2.5 U
        4C : 20 min incubation, 1 U
        
         Gel electrophoresis 8  -  Digestion conditions
          comparison 
        

          
        

         Samples were then checked using a Nanodrop 2000
          spectrophotometer. 
        

        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              1A after digestion
              121.4
              1.52
              0.29
            
            
              1B after digestion
              117.2
              1.52
              0.29
            
            
              1C after digestion
              118
              1.55
              0.29
            
             
              2A after digestion
              69.3
              1.64
              0.3
            
            
              2B after digestion
              116.5
              1.49
              0.3
            
            
              2C after digestion
              117.3
              1.51
              0.28
            
            
              3A after digestion
              119
              1.55
              0.28
            
            
              3B after digestion
              110.6
              1.54
              0.28
            
            
              3C after digestion
              117.4
              1.57
              0.24
            
            
              4A after digestion
              120.5
              1.52
              0.26
            
            
              4B after digestion
              118.3
              1.56
              0.24
            
             
              4C after digestion
              130.1
              1.52
              0.26
            
          
        
        

        
          Interpretations
        
        
          There was no bands on the gel. However, the nanodrop detected DNA with a pretty good 260/280 ratio. But there was no difference between the different conditions. So, either the single-strand DNA i s not revealed with SYBR Green or the digestion enzyme does not work properly.
        
        

        
          08.14.19
        
        
          Denaturation  -  of PCR 5 products
        
        
          In order to optimize the digestion step, we tried to denature our PCR product before being digested. Samples were heated at 95°C for 5 min and directly put in ice for 15 min.
        
        

        
          Digestion  -  Testing different conditions on PCR 5 products
        
        
          Different conditions of digestion were tested in order to optimize this step (buffers, DNA quantity to digest, enzyme quantity, with/without denaturation before).
          Conditions tested:
          
Cutsmart, 15 µg DNA, 1 U (37°C, 15 min)
          Cutsmart, 10 µg DNA, 1 U (37°C, 15 min)
          Cutsmart, 15 µg DNA, 0,5 U (37°C, 15 min)
          Cutsmart, 10 µg DNA, 0,5 U (37°C, 15 min)
          Lambda buffer, 15 µg DNA, 1 U (37°C, 15 min)
          Lambda buffer, 10 µg DNA, 1 U (37°C, 15 min)
          Lambda buffer, 15 µg DNA, 0,5 U (37°C, 15 min)
          Lambda buffer, 10 µg DNA, 0,5 U (37°C, 15 min)
          Lambda buffer, 15 µg DNA, 1 U (37°C, 15 min) + denaturation before
          Lambda buffer, 10 µg DNA, 1 U (37°C, 15 min) + denaturation before
          Lambda buffer, 15 µg DNA, 0,5 U (37°C, 15 min) + denaturation before
          Lambda buffer, 10 µg DNA, 0,5 U (37°C, 15 min) + denaturation before
          50 bp ladder
          PCR product before digestion
          PCR product before digestion followed by heat shock
        
        

        
          Gel electrophoresis 9  -  Digestion conditions comparison
        
        
          A 4% agarose gel was made to compare all the different conditions of digestion.
        
        
          
        
        

        
          Interpretations
        
        
          We are looking for a band with a shift in size compared to the PCR product before digestion. Once again, bands were similar to the PCR product, it means that conditions were too restrictive and that the digestion did not worked.
        
        

        
          08.15.19
        
        
          PCR 6  -  Generation of new materials
        
        
          To continue our experiments, a new part of the library was amplified in order to work with. Samples were also analyzed using a Nanodrop 2000 spectrophotometer.
        
        

        
          
            Sample
            Concentration (ng/μL)
            260/280 ratio
            260/230 ratio
          
          
            Sample 1
            1231.1
            1.65
            1.15
          
          
            Sample 2
            1217.1
            1.5
            1.15
          
          
            Sample 3
            1219.1
            1.66
            1.16
          
          
            Sample 4
            1215.3
            1.64
            1.15
          
          
            Sample 5
            1216.2
            1.65
            1.15
          
          
            Sample 6
            1213.2
            1.66
            1.14
          
          
            Sample 7
            1201.4
            1.66
            1.14
          
          
            Sample 8
            1211.5
            1.65
            1.15
          
          
            Sample 9
            1221.2
            1.65
            1.14
          
          
            Sample 10
            1185.2
            1.65
            1.14
          
        
        
      
      
        
          
          
          
          
        
        
          August Week 3  :  08/19  to
           08/21 

          

        
        08.19.19
        PCR 7 - Generation of new materials (10 ng, 5 ng, 1
          ng)
        A new PCR was performed to produce starting material. For
          this, different amounts of DNA to be amplified have been tested (10
          ng, 5 ng, 1 ng) in order to reduce nonspecific amplifications.
        

        
          
            
              

              
               Volume needed (in μL)
            
            
              Components
              Mix 1(10ng)
              Mix 2(5ng)
              Mix 3(1ng)
            
            
              Phusion Buffer 10X
              100
              100
              100
            
            
              dNTPs 25 mM
              10
              10
              10
            
            
              Forward primer (1/10)
              20
              20
              20
            
            
               Reverse primer (1/10)
              20
              20
              20
            
            
               Template DNA (1/1000)
              45
              22.5
              5
            
            
              Phusion DNA polymerase
              5
              5
              5
            
            
              Nuclease-free water
              300
              322.5
              340
            
            
              Total volume
              500
              500
              500
            
          
        
        Samples were also analyzed using a Nanodrop 2000
          spectrophotometer.
        
          
            
              Sample
              Concentration (ng/μl)
              260/280 ratio
              260/230 ratio
            
            
              Sample pooled (10ng)
              990.6
              1.62
              1.05
            
            
              Sample pooled (5ng)
              1028.5)
              1.61
              1.05
            
            
              Sample pooled (1ng)
              1028.7)
              1.61
              1.04
            
          
        
        08.20.10
         Digestion - 30 min, 5 U, from 5 to 1 µg with control
          without enzyme from PCR 7 
        A new digestion was performed with different quantity of
          DNA to digest for each sample. An undigested sample from the PCR 7 was
          used as a negative control.
        Gel electrophoresis 10 - Digestion conditions comparison
        
        A gel with 4% of agarose was made to compare each sample.
          The staining was performed with GelRed because it is more specific of
          single-strand DNA than ethidium bromide and SYBR Green.
         
         08.21.19 
         PCR 8 - Generation of new materials 
        A new PCR was performed to produce starting material. 

          Samples were analyzed using a Nanodrop 2000 spectrophotometer.
        
          
            
              Sample
              Concentration(ng/µl
              260/280 ratio
              260/230 ratio
            
            
              PCR 8 pooled
              1093.9
              1.63
              1.11
            
          
          
        
      
      
        
          
          
          
          
        
        
                   August Week 4  :   08/26  to
           09/1 
        

        

        

        

         08.26.19 
        

         Purification of PCR product  -  illustra
          MicroSpin G-25 Columns kit from GE Healthcare  -  PCR 9 
         The PCR 9 products were purified according to the
          manufacturer’s protocol. Then, samples were analyzed using a Nanodrop
          2000 spectrophotometer. 
        

        
          
            
              Sample
              Concentration (ng/μL)
              260/280 ratio
              260/230 ratio
            
            
              PCR 9 purified by column
              868.7
              1.58
              1.03
            
          
        
        

         Digestion  -  from PCR 9 
         After the purification, the sample was digested
          according to the manufacturer’s instructions with 4 µg of DNA. 
        

         Phenol/chloroform extraction  -  Digested
          products from PCR 9 
         The sample was purified by phenol/chloroform according
          to the protocol previously described. Then, samples were analyzed
          using a Nanodrop 2000 spectrophotometer. 
        

        

        
          
            
              Sample
              Concentration (ng/μL)
              260/280 ratio
              260/230 ration
            
            
              PCR 9 purified by phenol/chloroform
              17
              1.48
              1.34
            
          
        
        

         Interpretation 
         We lose a huge quantity of DNA. Thus, the concentration
          is too low to begin the first round of SELEX. 
        

         08.27.19 
        

         Digestion - from PCR 9 
         We purify the digested samples using either 2.5 X volume
          of ethanol, as previously, or 1 X volume of isopropanol. The samples
          were then analyzed using a Nanodrop 2000 spectrophotometer. 
        

        
          
            
              Sample
              Concentration (ng/μL)
              260/280 ratio
              260/230 ration
            
            
              PCR 9 purified with ethanol    1
              9.2
              1.6
              0.91
            
            
              PCR 9 purified with ethanol    2
              7.3
              1.5
              0.91
            
            
              PCR 9 purified with isopropanol    1
              44.9
              1.41
              1.10
            
            
              PCR 9 purified with isopropanol    1
              48.1
              1.42
              1.39
            
          
        
         Interpretations 
         The concentration of DNA are much higher when we used
          isopropanol compared to ethanol. 
        

         Conclusion 
         Isopropanol is used for the next purifications by
          phenol/chloroform. These concentrations are enough to perform the
          first round of SELEX on one bacteria. However, we want to select
          aptamers for two different bacteria so we must perform this step
          again. 
        

         08.28.19 
         Bacteria culture  -  E. faecium and S. aureus
        
         The strains E. faecium and S. aureus were grown on Petri
          dish on Blood agar and LB media respectively. The plates were
          incubated at 37°C during 24 hours. 
          
         Digestion  -  from PCR9 
         The PCR 9 products were digested using 4 µg of DNA
          according to the manufacturer’s protocol. 
         Phenol/chloroform extraction with Isopropanol
           -  Digested products from PCR 9 
         After digestion, the samples were purified using 1 X
          volume of isopropanol. The samples are then analysed using a Nanodrop
          2000 spectrophotometer. 
        

        
          
            
              Sample
              Concentration (ng/μL)
              260/280 ratio
              260/230 ration
            
            
              PCR 9 purified by phenol/chloroform  
                 1
              42.1
              1.46
              1.40
            
            
              PCR 9 purified by phenol/chloroform 2
              3.1
              1.65
              0.71
            
            
              PCR 9 purified by phenol/chloroform 3
              3.1
              1.55
              1.64
            
            
              PCR 9 purified by phenol/chloroform 4
              8.6
              1.43
              1.19
            
          
        
        

         Interpretations 
         The concentration of DNA is lower than expected. We may
          have pipeted some DNA as the pellet is invisible. 
        

         Conclusion 
         Because of these low concentrations, the first round of
          SELEX cannot be performed. 
        

         08.29.19 
        

         Bacteria culture  -  E. faecium and S. aureus
        
         Few colonies of E. faecium and S. aureus were taken from
          the Petri dish culture of the 28.08 and grow in their respective
          liquid media at 37°C under agitation. After 3 hours of growth, they
          are centrifuged and the pellet of bacteria is resuspended in PBS 1X.
          Dilutions were performed until the OD reached between 0.5 and 0.6. 
        

         Digestion  -  from PCR9 
         In order to have more DNA before starting the SELEX, we
          performed a digestion with 4 µg of DNA from the PCR 9 samples
          according to the manufacturer’s protocol. 
        

         Phenol/chloroform extraction with Isopropanol
           -  Digested products from PCR 9 
         The samples were purified after digestion using 1 X
          volume of isopropanol. Then, they were analysed with a Nanodrop 2000
          spectrophotometer. 
        

        
          
            Sample
            Concentration (ng/μL)
            260/280 ratio
            260/230 ration
          
          
            PCR 9 purified sample 1
            41.4
            1.41
            1.46
          
          
            PCR 9 purified sample 2
            4.2
            1.25
            0.57
          
          
            PCR 9 purified sample 3
            9.4
            1.33
            0.99
          
           
            PCR 9 purified sample 4
            23.7
            1.39
            1.08
          
          
            PCR 9 purified sample 5
            11.1
            1.37
            0.41
          
          
            PCR 9 purified sample 6
            27.8
            1.40
            0.90
          
        
        

        
          Conclusion
        
        

        
          After the purification, we have enough DNA concentration to start the SELEX on the two bacteria.
        
        
        
          SELEX 1 Round 1
        
        
          The first round of SELEX was performed  with 2 hours of incubation. The samples are then analyzed using a Nanodrop 2000 spectrophotometer.
        
        

        
          
            Sample
            Concentration (ng/μL)
            260/280 ratio
            260/230 ration
          
          
            S. aureus
            60.1
            1.61
            0.96
          
           
            E. faecium
            43.6
            1.69
            1.09
          
        
        

        
          PCR 10  -  Amplification of EFr1 & SAr1
        
        
          This PCR was performed to amplified the DNA after the first round of SELEX. We tried two different concentrations for both samples. The PCR followed the same program as described previously. It was run overnight and hold at 4°C.
        
        

        
          
            
            S aureus
          
          
            Components
            SA 1 (1/10)
            SA 2 (1/90)
          
          
            Phusion buffer
            10
            10
          
          
            dNTPs
            1
            1
          
          
            Forward primer
            2
            2
          
          
            Reverse primer
            2
            2
          
          
            Template DNA (1/10)
            1
            0
          
          
            Template DNA (1/90)
            0
            1
          
          
            Phusion DNA polymerase
            0.5
            0.5
          
           
            Nulease-free water
            33.5
            33.5
          
          
            Total volume
            50
            50
          
        
        



        
          
            
            E. faecium
          
          
            Components
            SA 1 (1/10)
            SA 2 (1/80)
          
          
            Phusion buffer
            10
            10
          
          
            dNTPs
            1
            1
          
          
            Forward primer
            2
            2
          
          
            Reverse primer
            2
            2
          
          
            Template DNA (1/10)
            1
            0
          
          
            Template DNA (1/80)
            0
            1
          
          
            Phusion DNA polymerase
            0.5
            0.5
          
           
            Nulease-free water
            33.5
            33.5
          
          
            Total volume
            50
            50
          
        
        

        
          The samples are analyzed using a Nanodrop 2000 spectrophotometer.
        
        


        
          
            Sample
            Concentration (ng/μL)
            260/280 ratio
            260/230 ration
          
          
            PCR 10 - Round 1 - SA    1
            1253.9
            1.62
            1.05
          
           
            PCR 10 - Round 1 - SA    2
            1200.4
            1.64
            1.09
          
          
            PCR 10 - Round 1 - EF    1
            1118.9
            1.56
            0.89
          
          
            PCR 10 - Round 1 - EF    2
            1207.9
            1.66
            1.13
          
        
        

        
          08.30.19
        
        

        
          Gel electrophoresis 12  -  PCR 10
        
        
          On a 4% agarose gel, samples before and after the PCR 12 were run.
        
        
      
      
        
          
          
          
          
        
        
           
        
      
      
        
          
          
          
          
        
        
                   
          September Week 1  :  09/05  to  09/15
        
        

        
          Digestion   -  EFr1 & SAr1
        
        
          The digestion was performed with 4 µg of DNA according to the manufacturer’s protocol.
        
        

        
          
            Sample
            Concentration (ng/μL)
            260/280 ratio
            260/230 ratio
          
          
            EFr1 digested
            27.4
            1.87
            0.26
          
           
            SAr1 digested
            30.2
            1.89
            0.28
          
        
        

        
        Purification of digested products EFr1 & SAr1  -  kit Monarch from NEB
        
        
          A new kit of purification was tried to purify the PCR Products. The experiment was performed according to the manufacturer’s recommendations.
        
        

        
          The samples are checked using a Nanodrop 2000 spectrophotometer.
        
        

        
          
            Sample
            Concentration (ng/µl)
            260/280 ratio
            260/230 ratio
          
          
            EFr1 purified
            7.8
            1.27
            0.27
          
          
            EFr1 wash 1
            -1.7
            0.58
            -0.03
          
          
            EFr1 wash 2
            2.5
            5.51
            0.27
          
          
            EFr1 purified
            7.8
            1.27
            0.27
          
          
            SAr1 purified
            7.2
            1.52
            0.58
          
          
            SAr1 wash 1
            22.5
            1.29
            0.53
          
          
            SAr1 wash 2
            2.4
            8.31
            0.30
          
        
        

        
          Interpretations
        
        
          The concentrations are really low. Moreover, the quantity of DNA within the sample is not high because the ratio 260/280 is not close to 2. Finally, the ratio 260/230 did not change increase before and after the purification.
        
        

        
          Conclusion
        
        
          This kit cannot be used to purify our DNA after digestion.
        
        

        
          Purification of digested products EFr1 & SAr1  -   illustra MicroSpin G-25 Columns kit from GE Healthcare
        
        

        
          The purification was performed according to the manufacturer’s protocol.
The samples are checked using a Nanodrop 2000 spectrophotometer.
        
        

        
          
          	Sample
            Concentration (ng/μL)
            260/280 ratio
            260/230 ratio
          
          
          	EFr1 purified
            53.2
            1.62
            0.24
          
          
          	SAr1 purified
            56.2
            1.54
            0.27
          
        
        

        
          Interpretation
        
        
          Even if the concentrations are quite elevated, we noticed that the ratio 260/230 did not change before and after the purification using these columns. Hence, the enzymes may not be  purified by this technique.
        
        

        
          Conclusion
        
        
          We tried several kits to purify our PCR products but none of them worked. Thus, we decided to directly continue with the digestion after the PCR without purifying. All the enzymes will be removed during the phenol/chloroform extraction just before the SELEX.
        
        

        

        
          09.06.19
        
        

        
          Bacteria culture  -  E. faecium and S. aureus
        
        
          Few colonies of E. faecium and S. aureus were taken from the Petri dish culture of the 28.08 and grow in their respective liquid media at 37°C under agitation. After 3 hours of growth, they are centrifuged and the pellet of bacteria is resuspended in PBS 1X. Dilutions were performed until the OD reached between 0.5 and 0.6.
        
        

        
          Phenol/chloroform extraction with Isopropanol  -  Digested products EFr1 & SAr1
        
        
          The samples were purified after digestion. Then, they were analysed with a Nanodrop 2000 spectrophotometer.
        
        

        
          
            Sample
            Concetration (ng/μL)
            260/280 ratio
            260/230 ratio
          
          
            EFr1 sample 1
            17.2
            1.46
            1.29
          
           
            EFr1 sample 2
            26.5
            1.49
            1.44
          
          
            EFr1 sample 3
            29.8
            1.49
            1.47
          
          
            EFr1 sample 4
            37.2
            1.56
            1.5
          
          
            SAr1 sample 1
            69
            1.51
            1.54
          
          
            SAr1 sample 2
            61.5
            1.75
            0.28
          
          
        
        
          Interpretations
        
        
          A total quantity of 1728 pmol was obtained for E. faecium in the Round 1, and 869 pmol for S. aureus.
        
        

        
          SELEX Round 2
        
        
          The first round of SELEX was performed  with 2 hours of incubation. The samples are then analyzed using a Nanodrop 2000 spectrophotometer.
        
        

        
          
            Sample
            Concetration (ng/μL)
            260/280 ratio
            260/230 ratio
          
          
            SAr2
            33.8
            2.19
            1.45
          
          
            EFr2
            32
            2.21
            1.6
          
        
        


        
          PCR 10  -  Amplification of EFr2 and SAr2
        
        
          This PCR was performed to amplified the DNA after the second round of SELEX. The PCR followed the same program as described previously. It was run overnight and hold at 4°C.
        
        


        
          
            Components
            SAr1
            EFr1
          
          
            Phusion buffer
            200
            200
          
          
            dNTPs
            20
            20
          
          
            Forward primer
            40
            40
          
           
            Reverse primer
            40
            40
          
          
            Template DNA (1/3)
            20
            20
          
           
            Phusion DNA polymerase
            10
            10
          
          
            Nuclease-free water
            670
            670
          
          
            Total volume
            1000
            1000
          
        
        

        
          The samples are analyzed using a Nanodrop 2000 spectrophotometer.
        
        

        
          
            Sample
            Concetration (ng/μL)
            260/280 ratio
            260/230 ratio
          
          
            PCR 10 - Round 2 - SA
            1086.6
            1.67
            1.20
          
          
            PCR 10 - Round 2 - EF
            1243.4
            1.66
            1.20
          
        
        
      
      
        
          
          
          
          
        
        
           

        
      
      
        
          
          
          
          
        
        
            September Week 3  :  09/16
           to  09/21 
         09.16.19 
        Bacteria culture - E. faecium and S. aureus
        
         E. faecium and S. aureus were grown
          until the OD reached between 0.5 and 0.6
         Digestion - EFr5 & SAr5 
         PCR samples from the Round 5 of SELEX were digested
          using lambda exonuclease. A mix was performed in order to scale-up
          this step and get enough digested material. Samples concentrations
          were checked using a Nanodrop 2000 spectrophotometer. 
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              Dig - Round 5 - SA sample 1
              56.7
              1.75
              0.25
            
            
              Dig - Round 5 - SA sample 2
              57.1
              1.79
              0.23
            
            
              Dig - Round 5 - EF sample 1
              57.6
              1.78
              0.25
            
            
               Dig - Round 5 - EF sample 2
              57.6
              1.81
              0.23
            
          
        
        

        Phenol/chloroform extraction with Isopropanol and
          Glycogen - Digested products EFr5 & SAr5
         Once digested, samples were purified after digestion.
          They were analysed with a Nanodrop 2000 spectrophotometer. 
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              EFr5 sample 1
              24.5
              1.67
              0.94
            
            
              EFr5 sample 2
              21.1
              1.67
              0.98
            
            
              SAr5 sample 1
              104.2
              1.50
              1.56
            
            
              SAr5 sample 2
              160
              1.54
              1.61
            
          
        
        

        Interpretation
        A total quantity of 354 pmol was obtained for E.
            faecium in the Round 5, and 2040 pmol for S. aureus. 
         SELEX Round 6 
         The Round 6 of SELEX was performed by incubating for 1h.
          The samples are then analyzed using a Nanodrop 2000 spectrophotometer.
        
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              SAr6
              45.3
              1.91
              1.33
            
            
              EFr6
              23.7
              2.22
              1.91
            
          
        
        

        PCR 14 - Amplification of EFr6 and SAr6
        This PCR was performed to amplified the DNA after the
          Round 6 of SELEX. The PCR followed the same program as described
          previously with the same concentration. It was run overnight and hold
          at 4°C. The samples are analyzed using a Nanodrop 2000
          spectrophotometer. 
        
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              PCR 14 - Round 6 - SA
              1616.9
              1.65
              1.20
            
            
              PCR 14 - Round 6 - EF
              2068.6
              1.70
              1.37
            
          
        
        

         09.17.19 
         Bacteria culture - E. faecium and S. aureus
        
         E. faecium and S. aureus were grown
          until the OD reached between 0.5 and 0.6 
         Digestion - EFr6 & SAr6 
         PCR samples from the Round 6 of SELEX were digested
          using lambda exonuclease. A mix was performed in order to scale-up
          this step and get enough digested material. Samples concentrations
          were checked using a Nanodrop 2000 spectrophotometer. 
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              Dig - Round 6 - SA sample 1
              57.5
              1.78
              0.24
            
            
              Dig - Round 6 - SA sample 2
              56.1
              1.81
              0.26
            
          
          
            
              Dig - Round 6 - EF sample 1
              62.4
              1.82
              0.28
            
          
          
            
              Dig - Round 6 - EF sample 2
              58.9
              1.89
              0.24
            
          
        
         Phenol/chloroform extraction with Isopropanol and
          Glycogen - Digested products EFr6 & SAr6 
         Once digested, samples were purified after digestion.
          They were analysed with a Nanodrop 2000 spectrophotometer. 
        

        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              EFr6 sample 1
              245
              1.65
              1.73
            
            
              EFr6 sample 2
              68.4
              1.47
              1.30
            
            
              SAr6 sample 1
              19.6
              1.52
              1.19
            
            
              SAr6 sample 2
              15.9
              1.60
              1.00
            
          
        
        

         Interpretation 
         A total quantity of 2440 pmol was obtained for E.
            faecium the Round 6, and 276 pmol for S. aureus. 
         SELEX Round 7 
         The Round 7 of SELEX was performed by incubating for 1h.
          The samples are then analyzed using a Nanodrop 2000 spectrophotometer.
        
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              SAr7
              25.4
              2.18
              1.41
            
            
              EFr7
              37.7
              2.08
              1.93
            
          
        
        

         PCR 15 - Amplification of EFr7 and SAr7 
         This PCR was performed to amplified the DNA after the
          Round 7 of SELEX. The PCR followed the same program as described
          previously with the same concentration. It was run overnight and hold
          at 4°C. The samples are analyzed using a Nanodrop 2000
          spectrophotometer. 
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              PCR 15 - Round 7 - SA
              1358
              1.62
              1.05
            
            
              PCR 15 - Round 7 - EF
              1572.8
              1.65
              1.18
            
          
        
        

        09.18.19
         Bacteria culture - E. faecium and S. aureus
        
         E. faecium and S. aureus were grown
          until the OD reached between 0.5 and 0.6. 
        Digestion - EFr7 & SAr7 
         PCR samples from the Round 7 of SELEX were digested
          using lambda exonuclease. A mix was performed in order to scale-up
          this step and get enough digested material. 

          Samples concentrations were checked using a Nanodrop 2000
          spectrophotometer. 
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              Dig - Round 7 - SA sample 1
              50.3
              1.81
              0.25
            
            
              Dig - Round 7 - SA sample 2
              58.1
              1.78
              1.24
            
            
              Dig - Round 7 - EF sample 1
              60.4
              1.83
              0.24
            
            
              Dig - Round 7 - EF sample 2
              59.9
              1.81
              0.28
            
          
        
        

         Phenol/chloroform extraction with Isopropanol and
          Glycogen - Digested products EFr7 & SAr7 
         Once digested, samples were purified after digestion.
          They were analysed with a Nanodrop 2000 spectrophotometer. 
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              EFr7 sample 1
              29.8
              1.52
              1.20
            
            
              EFr7 sample 2
              13.4
              1.47
              1.10
            
            
              SAr7 sample 1
              98.6
              1.52
              1.55
            
            
              SAr7 sample 2
              247.7
              1.72
              1.79
            
          
        
        

         Interpretation 
         A total quantity of 336 pmol was obtained for E.
            faecium in the Round 7, and 1340 pmol for S. aureus. 
         SELEX Round 8 
         The Round 8 of SELEX was performed by incubating for 1h.
          The samples are then analyzed using a Nanodrop 2000 spectrophotometer.
        
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              Sar8
              19.3
              2.44
              1.57
            
            
              EFr8
              17.2
              2.30
              1.33
            
          
        
        

         PCR 16 - Amplification of EFr8 and SAr8 
         This PCR was performed to amplified the DNA after the
          Round 8 of SELEX. The PCR followed the same program as described
          previously with the same concentration. It was run overnight and hold
          at 4°C. The samples are analyzed using a Nanodrop 2000
          spectrophotometer. 
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              PCR 16 - Round 8 - SA
              933.2
              1.58
              0.93
            
            
              PCR 16 - Round 8 - EF
              1324.2
              1.60
              0.98
            
          
        
        

         09.19.19 
        Bacteria culture - E. faecium and S. aureus 
         PCR samples from the Round 8 of SELEX were digested
          using lambda exonuclease. A mix was performed in order to scale-up
          this step and get enough digested material. 

          Samples concentrations were checked using a Nanodrop 2000
          spectrophotometer.
         Digestion - EFr8 & SAr8 
         PCR samples from the Round 8 of SELEX were digested
          using lambda exonuclease. A mix was performed in order to scale-up
          this step and get enough digested material. Samples concentrations
          were checked using a Nanodrop 2000 spectrophotometer. 
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              Dig - Round 8 - SA sample 1
              55.5 
              1.78
              0.22
            
            
              Dig - Round 8 - SA sample 2
              54.2
              1.79
              0.23
            
            
              Dig - Round 8 - EF sample 1
              46
              1.77
              0.23
            
            
              Dig - Round 8 - EF sample 2
              44.5
              1.75
              1.23
            
          
        
         09.20.19
         Phenol/chloroform extraction with Isopropanol and
          Glycogen - Digested products EFr8 & SAr8 
         Once digested, samples were purified after digestion.
          They were analysed with a Nanodrop 2000 spectrophotometer. 
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              EFr8 sample 1
              100.4
              1.53
              1.49
            
            
              EFr8 sample 2
              123.3
              1.56
              1.59
            
            
              SAr8 sample 1
              123.3
              1.56
              1.59
            
            
              SAr8 sample 2
              160.5
              1.58
              1.75
            
          
        
         SELEX Round 9 
         The Round 9 of SELEX was performed by incubating for 45
          min. The samples are then analyzed using a Nanodrop 2000
          spectrophotometer. 
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              SAr9
              28.1
              2.20
              1.47
            
            
              SAr8
              18.2
              2.32
              1.73
            
          
        
        PCR 17 - Amplification of EFr9 and SAr9 
         This PCR was performed to amplified the DNA after the
          Round 9 of SELEX. The PCR followed the same program as described
          previously with the same concentration. It was run overnight and hold
          at 4°C. The samples are analyzed using a Nanodrop 2000
          spectrophotometer. 
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              PCR 17 - Round 9 - SA
              541.4
              1.47
              0.74
            
            
              PCR 17 - Round 9 - EF
              947.6
              1.59
              0.96
            
          
        
         Transformation - biobrick BBa_J04450 (mRFP) 
         As instructed by iGEM, the biobrick was resuspended with
          10 µL of TE buffer. Then, it was transferred into a tube containing
          100 µL of E. coli DH5α competent cells and let 30 minutes in
          ice. Then, the sample was heated at 42°C for 40 seconds and, after,
          put in ice for 3 minutes. Therefore, 600 µL of SOC media was added and
          the sample was incubated at 37°C during 1 hour under agitation at 140
          rpm. Then, the bacteria are put on a LB petri dish containing
          kanamycin at 25 µg/mL and stored all night at 37°C 
         09.21.19 
         Bacteria culture - E. faecium and S. aureus 
         E. faecium and S. aureus were grown
          until the OD reached between 0.5 and 0.6
         Digestion - EFr9 & SAr9
         PCR samples from the Round 9 of SELEX were digested
          using lambda exonuclease. A mix was performed in order to scale-up
          this step and get enough digested material. 

          Samples concentrations were checked using a Nanodrop 2000
          spectrophotometer. 
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              Dig - Round 9 - SA sample 1
              59.3
              1.67
              0.21
            
            
              Dig - Round 9 - SA sample 2
              60.1
              1.69
              0.18
            
            
              Dig - Round 9 - EF sample 1
              56
              1.77
              0.23
            
            
              Dig - Round 9 - EF sample 2
              54.9
              1.77
              0.23
            
          
        
         Phenol/chloroform extraction with Isopropanol and
          Glycogen - Digested products EFr9 & SAr9 
         Once digested, samples were purified after digestion.
          They were analysed with a Nanodrop 2000 spectrophotometer. 
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              EFr9 sample 1
              90.8
              1.52
              1.52
            
            
              EFr9 sample 2
              6124
              1.54
              1.64
            
            
              SAr9 sample 1
              203.9
              1.57
              1.69
            
            
              SAr9 sample 2
              315.1
              1.71
              1.70
            
          
        
         SELEX Round 10 
         The Round 10 of SELEX was performed by incubating for 45
          min. The samples are then analyzed using a Nanodrop 2000
          spectrophotometer. 
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              SAr10
              20.8
              2.33
              1.56
            
            
              EFr10
              20.4
              2.54
              1.71
            
          
        
         PCR 18 - Amplification of EFr10 and SAr10 
         This PCR was performed to amplified the DNA after the
          Round 10 of SELEX. The PCR followed the same program as described
          previously with the same concentration. It was run overnight and hold
          at 4°C. Samples were analyzed using a Nanodrop 2000 spectrophotometer.
        
        
          
            
              Sample
              Concentration (ng/µl)
              260/280 ratio
              260/230 ratio
            
            
              PCR 18 - Round 10 - SA
              1123.6
              1.64
              1.13
            
            
              PCR 18 - Round 10 - EF
              560.7
              1.46
              0.73

@@ Line 715: / Line 715: @@
          </div>
          <div id="JU2" class="ent"  >
+            <p class="p1N"> July Week 2 &nbsp;: &nbsp;09.09.19 &nbsp;to
+          &nbsp; 09.13.19<br>
+        </p>
+        <p class="date">09.09.19</p>
+        <p class="p3N">Bacteria culture - <i>E. coli</i> containing the plasmid
+          PET28:GFP </p>
+        <p class="p2N">In order to extract the plasmid PET28:GFP, <i>E. coli</i>
+          was cultured on LB petri dishes containing kanamycin at 50 µg/mL. The
+          cultures were stored all night at 37°C. </p>
+        <p class="p3N">Bacteria culture - <i>E. faecium</i> and <i>S. aureus</i></p>
+        <p class="p2N"><i>E. faecium</i> and <i>S. aureus</i> were grown until
+          the OD reached between 0.5 and 0.6.</p>
+        <p class="p3N"> Digestion - EFr2 &amp; SAr2</p>
+        <p class="p2N">PCR samples from the Round 2 of SELEX were digested using
+          lambda exonuclease. A mix was performed in order to scale-up this step
+          and get enough digested material. <br>
+        </p>
+        <p class="p2N" ,="" style="margin-left:50px">1. Prepare the mix as
+          follows</p>
+        <table class="notTABLE">
+          <tbody>
+            <tr class="NotTR">
+              <td class="notTD"><br>
+              </td>
+              <td class="notTD">50µL reaction</td>
+              <td class="notTD">Mix</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">DNA (PCR11)</td>
+              <td class="notTD">4 µg</td>
+              <td class="notTD">120 µg</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">Lambda exonuclease buffer (10X)</td>
+              <td class="notTD">5µL</td>
+              <td class="notTD">µL</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">Lambda exonuclease</td>
+              <td class="notTD">1µL</td>
+              <td class="notTD">30µL</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">Nuclease-free H²0</td>
+              <td class="notTD">QSP 50 µL</td>
+              <td class="notTD">QSP 1.5 mL</td>
+            </tr>
+          </tbody>
+        </table>
+        <br>
+        <p class="p2N" ,="" style="margin-left:50px">2. Incubate samples for 30
+          min at 37°C in a water-bath</p>
+        <p class="p2N" ,="" style="margin-left:50px">3. Stop the reaction by
+          adding EDTA to 10 mM</p>
+        <p class="p2N" ,="" style="margin-left:50px">4. Heat at 75°C for 10
+          minutes to inactivate the enzyme</p>
+        <p class="p2N">Samples concentrations were checked using a Nanodrop 2000
+          spectrophotometer.</p>
+        <table class="notTABLE">
+          <tbody>
+            <tr class="NotTR">
+              <td class="notTD">Sample</td>
+              <td class="notTD">Concentration (ng/µl)</td>
+              <td class="notTD">260/280 ratio</td>
+              <td class="notTD">260/230 ratio</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">Dig-Round 2 - SA</td>
+              <td class="notTD">32.6</td>
+              <td class="notTD">1.91</td>
+              <td class="notTD">0.27</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">Dig - Round 2- EF</td>
+              <td class="notTD">20</td>
+              <td class="notTD">1.92</td>
+              <td class="notTD">0.25</td>
+            </tr>
+          </tbody>
+        </table>
+        <br>
+        <p class="p3N">Phenol/chloroform extraction with Isopropanol - Digested
+          products EFr2 &amp; SAr2<!--^--> </p>
+        <p class="p2N">Once digested, samples were purified after digestion.
+          They were analysed with a Nanodrop 2000 spectrophotometer.</p>
+        <table class="notTABLE">
+          <tbody>
+            <tr class="NotTR">
+              <td class="notTD">Sample</td>
+              <td class="notTD">Concentration (ng/µl)</td>
+              <td class="notTD">260/280 ratio</td>
+              <td class="notTD">260/230 ratio</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">EFr2 sample 1</td>
+              <td class="notTD">46.7</td>
+              <td class="notTD">1.47</td>
+              <td class="notTD">1.49</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">EFr2 sample 2</td>
+              <td class="notTD">42.1</td>
+              <td class="notTD">1.48</td>
+              <td class="notTD">1.52</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">SAr2 sample 1</td>
+              <td class="notTD">29.1</td>
+              <td class="notTD">1.66</td>
+              <td class="notTD">1.63</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">SAr2 sample 2</td>
+              <td class="notTD">37.3</td>
+              <td class="notTD">1.52</td>
+              <td class="notTD">1.55</td>
+            </tr>
+          </tbody>
+        </table>
+        <br>
+        <p class="p3N">Interpretation</p>
+        <p class="p2N">A total quantity of 692 pmol was obtained for <i>E.
+            faecium</i> in the Round 2, and 516 pmol for <i>S. aureus</i></p>
+        <p class="p3N">SELEX Round 3</p>
+        <p class="p2N">The first round of SELEX was performed with 1h45 hours of
+          incubation. The samples are then analyzed using a Nanodrop 2000
+          spectrophotometer.</p>
+        <table class="notTABLE">
+          <tbody>
+            <tr class="NotTR">
+              <td class="notTD">Sample</td>
+              <td class="notTD">Concentration (ng/µl)</td>
+              <td class="notTD">260/280 ratio</td>
+              <td class="notTD">260/230 ratio</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">SAr3</td>
+              <td class="notTD">33.6</td>
+              <td class="notTD">2.19</td>
+              <td class="notTD">1.45</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">EFr3</td>
+              <td class="notTD">42.9</td>
+              <td class="notTD">2.3</td>
+              <td class="notTD">1.76</td>
+            </tr>
+          </tbody>
+        </table>
+        <p class="p3N">PCR 11 - Amplification of EFr3 and SAr3</p>
+        <p class="p2N"> This PCR was performed to amplified the DNA after the
+          Round 3 of SELEX. The PCR followed the same program as described
+          previously with the same concentration. It was run overnight and hold
+          at 4°C. The samples are analyzed using a Nanodrop 2000
+          spectrophotometer. </p>
+        <table class="notTABLE">
+          <tbody>
+            <tr class="NotTR">
+              <td class="notTD">Sample</td>
+              <td class="notTD">Concentration (ng/µl)</td>
+              <td class="notTD">260/280 ratio</td>
+              <td class="notTD">260/230 ratio</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">PCR 11 - Round 3 - SA</td>
+              <td class="notTD">1086.6</td>
+              <td class="notTD">1.67</td>
+              <td class="notTD">1.20</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">PCR 11 - Round 3 - EF</td>
+              <td class="notTD">1496</td>
+              <td class="notTD">1.72</td>
+              <td class="notTD">1.38</td>
+            </tr>
+          </tbody>
+        </table>
+        <br>
+        <p class="date">09.10.19</p>
+        <p class="p3N">Bacteria culture - <i>E. coli</i> containing the plasmid
+          PET28:GFP</p>
+        <p class="p2N"> After an overnight growth, a colony of bacteria from the
+.09 was added into an erlenmeyer containing LB media. Then, the
+          erlenmeyer was put at 37°C all night under agitation at 140 rpm. </p>
+        <p class="p3N">Bacteria culture - <i>E. faecium</i> and <i>S. aureus</i></p>
+        <p class="p2N"><i>E. faecium</i> and <i>S. aureus</i> were grown until
+          the OD reached between 0.5 and 0.6 </p>
+        <p class="p3N">Digestion - EFr3 &amp; SAr3</p>
+        <p class="p2N"> PCR samples from the Round 3 of SELEX were digested
+          using lambda exonuclease. A mix was performed in order to scale-up
+          this step and get enough digested material. Samples concentrations
+          were checked using a Nanodrop 2000 spectrophotometer. </p>
+        <table class="notTABLE">
+          <tbody>
+            <tr class="NotTR">
+              <td class="notTD">Sample</td>
+              <td class="notTD">Concentration (ng/µl)</td>
+              <td class="notTD">260/280 ratio</td>
+              <td class="notTD">260/230 ratio</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">Dig - Round 3 - SA</td>
+              <td class="notTD">36.8</td>
+              <td class="notTD">1.92</td>
+              <td class="notTD">0.27</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">Dig - Round 3 - EF</td>
+              <td class="notTD">45.3</td>
+              <td class="notTD">1.81</td>
+              <td class="notTD">0.23</td>
+            </tr>
+          </tbody>
+        </table>
+        <br>
+        <p class="p3N"> Phenol/chloroform extraction with Isopropanol and
+          Glycogen - Digested products EFr3 &amp; SAr3 </p>
+        <p class="p2N"> Once digested, samples were purified after digestion
+          using a phenol/chloroform extraction. Glycogen for a final 0.5 μg/μL
+          concentration was added in order to maximise the amount of DNA
+          recovered and avoid pipetting the pellet. They were analysed with a
+          Nanodrop 2000 spectrophotometer. </p>
+        <table class="notTABLE">
+          <tbody>
+            <tr class="NotTR">
+              <td class="notTD">Sample</td>
+              <td class="notTD">Concentration (ng/µl)</td>
+              <td class="notTD">260/280 ratio</td>
+              <td class="notTD">260/230 ratio</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">EFr3 sample 1</td>
+              <td class="notTD">32.1</td>
+              <td class="notTD">1.56</td>
+              <td class="notTD">1.86</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">EFr3 sample 2</td>
+              <td class="notTD">22.7</td>
+              <td class="notTD">1.48</td>
+              <td class="notTD">1.89</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">SAr3 sample 1</td>
+              <td class="notTD">27.1</td>
+              <td class="notTD">1.52</td>
+              <td class="notTD">1</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">SAr3 sample 2</td>
+              <td class="notTD">65.4</td>
+              <td class="notTD">1.50</td>
+              <td class="notTD">1.24</td>
+            </tr>
+          </tbody>
+        </table>
+        <p class="p3N">Interpretation</p>
+        <p class="p2N"> A total quantity of 856 pmol was obtained for <i>E.
+            faecium</i> in the Round 3, and 516 pmol for <i>S. aureus</i>.
+          Because the purity of SAr3 sample 1 was really low, we only used SAr3
+          sample 2 for the following steps. </p>
+        <p class="date"> 09.11.19 </p>
+        <p class="p3N">Midiprep - <i>E. coli</i> containing the plasmid
+          PET28:GFP </p>
+        <p class="p2N"> The liquid culture of <i>E. coli</i> from the 10.09 was
+          centrifuged 10 minutes at 3,500 x g and the supernatant was removed.
+          Then, we used a midiprep kit from Qiagen to extract the plasmid from <i>E.
+            coli</i>. For that, the pellet was resuspended with 4 mL of P1
+          containing RNAse. Then, 4 mL of P2 was added and we waited 5 minutes
+          at RT. After that, 4 mL of P3 was added and the solution was mixed.
+          The solution was loaded on a column and the filtrate was recovered.
+          After, 4 mL of QBT Qiagen lip was added on the column and then the
+          filtrate was loaded on the column. After filtration, the column was
+          washed twice with QC buffer. In the filtrate, 5 mL of QF buffer and
+.5 mL of isopropanol were added. The solution was centrifuged during
+minutes at 15000 x g. The supernatant was removed and the pellet
+          was washed with 1 mL of ethanol 70% and centrifuged again 30 minutes
+          at 15,000 x g. We removed all the supernatant and let the pellet dry.
+          Then, 10 µL of TE buffer was added and the sample was vortexed.
+          Finally, the sample was stored at -20°C. The sample was checked with a
+          Nanodrop 2000 spectrophotometer. </p>
+        <table class="notTABLE">
+          <tbody>
+            <tr class="NotTR">
+              <td class="notTD">Sample</td>
+              <td class="notTD">Concentration (ng/µl)</td>
+              <td class="notTD">260/280 ratio</td>
+              <td class="notTD">260/230 ratio</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">pET28:GFP</td>
+              <td class="notTD">212.6</td>
+              <td class="notTD">1.88</td>
+              <td class="notTD">2.24</td>
+            </tr>
+          </tbody>
+        </table>
+        <br>
+        <p class="date">09.12.19 </p>
+        <p class="p3N"> Bacteria culture - <i>E. faecium</i> and <i>S. aureus</i>
+        </p>
+        <p class="p2N"><i>E. faecium</i> and <i>S. aureus</i> were grown until
+          the OD reached between 0.5 and 0.6</p>
+        <p class="p3N">SELEX Round 4</p>
+        <p class="p2N"> The Round 4 of SELEX was performed by incubating for
+h30. The samples are then analyzed using a Nanodrop 2000
+          spectrophotometer. </p>
+        <table class="notTABLE">
+          <tbody>
+            <tr class="NotTR">
+              <td class="notTD">Sample</td>
+              <td class="notTD">Concentration (ng/µl)</td>
+              <td class="notTD">260/280 ratio</td>
+              <td class="notTD">260/230 ratio</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">SAr4</td>
+              <td class="notTD">37.1</td>
+              <td class="notTD">2.02</td>
+              <td class="notTD">1.40</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">EFr4</td>
+              <td class="notTD">21.2</td>
+              <td class="notTD">2.25</td>
+              <td class="notTD">1.55</td>
+            </tr>
+          </tbody>
+        </table>
+        <br>
+        <p class="p3N"> PCR 12 - Amplification of EFr4 and SAr4 </p>
+        <p class="p2N"> This PCR was performed to amplified the DNA after the
+          Round 4 of SELEX. The PCR followed the same program as described
+          previously with the same concentration. It was run overnight and hold
+          at 4°C. The samples are analyzed using a Nanodrop 2000
+          spectrophotometer. </p>
+        <br>
+        <table class="notTABLE">
+          <tbody>
+            <tr class="NotTR">
+              <td class="notTD">Sample</td>
+              <td class="notTD">Concentration (ng/µl)</td>
+              <td class="notTD">260/280 ratio</td>
+              <td class="notTD">260/230 ratio</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">PCR 12 - Round 4 - SA</td>
+              <td class="notTD">1373.9</td>
+              <td class="notTD">1.62</td>
+              <td class="notTD">1.05</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">PCR 12 - Round 4 - EF</td>
+              <td class="notTD">1314.2</td>
+              <td class="notTD">1.62</td>
+              <td class="notTD">1.06</td>
+            </tr>
+          </tbody>
+        </table>
+        <br>
+        <p class="date"> 09.13.19 </p>
+        <p class="p3N">Bacteria culture - <i>E. faecium</i> and <i>S. aureus</i></p>
+        <p class="p2N"> <i>E. faecium</i> and <i>S. aureus</i> were grown
+          until the OD reached between 0.5 and 0.6 </p>
+        <p class="p3N"> Digestion - EFr4 &amp; SAr4 </p>
+        <p class="p2N"> PCR samples from the Round 4 of SELEX were digested
+          using lambda exonuclease. A mix was performed in order to scale-up
+          this step and get enough digested material. Samples concentrations
+          were checked using a Nanodrop 2000 spectrophotometer. </p>
+        <br>
+        <table class="notTABLE">
+          <tbody>
+            <tr class="NotTR">
+              <td class="notTD">Sample</td>
+              <td class="notTD">Concentration (ng/µl)</td>
+              <td class="notTD">260/280 ratio</td>
+              <td class="notTD">260/230 ratio</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">Dig - Round 4 - SA sample 1</td>
+              <td class="notTD">59.7</td>
+              <td class="notTD">1.79</td>
+              <td class="notTD">0.27</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">Dig - Round 4 - SA sample 2</td>
+              <td class="notTD">59.2</td>
+              <td class="notTD">1.80</td>
+              <td class="notTD">0.25</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">Dig - Round 4 - EF sample 1</td>
+              <td class="notTD">57.4</td>
+              <td class="notTD">1.80</td>
+              <td class="notTD">0.24</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">Dig - Round 4 - EF sample 2</td>
+              <td class="notTD">58.8</td>
+              <td class="notTD">1.79</td>
+              <td class="notTD">0.24</td>
+            </tr>
+          </tbody>
+        </table>
+        <br>
+        <p class="p3N"> Phenol/chloroform extraction with Isopropanol and
+          Glycogen - Digested products EFr4 &amp; SAr4 </p>
+        <p class="p2N"> Once digested, samples were purified after digestion.
+          They were analysed with a Nanodrop 2000 spectrophotometer. </p>
+        <table class="notTABLE">
+          <tbody>
+            <tr class="NotTR">
+              <td class="notTD">Sample</td>
+              <td class="notTD">Concentration (ng/µl)</td>
+              <td class="notTD">260/280 ratio</td>
+              <td class="notTD">260/230 ratio</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">EFr4 sample 1</td>
+              <td class="notTD">49.6</td>
+              <td class="notTD">1.46</td>
+              <td class="notTD">1.17</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">EFr4 sample 2</td>
+              <td class="notTD">34.1</td>
+              <td class="notTD">1.51</td>
+              <td class="notTD">1.13</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">SAr4 sample 1</td>
+              <td class="notTD">39.6</td>
+              <td class="notTD">1.54</td>
+              <td class="notTD">1.15</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">SAr4 sample 2</td>
+              <td class="notTD">18.7</td>
+              <td class="notTD">1.54</td>
+              <td class="notTD">1.33</td>
+            </tr>
+          </tbody>
+        </table>
+        <br>
+        <p class="p3N"> Interpretation </p>
+        <p class="p2N"> A total quantity of 650 pmol was obtained for <i>E.
+            faecium</i> in the Round 4, and 684 pmol for <i>S. aureus</i>.</p>
+        <p class="p3N"> SELEX Round 5 </p>
+        <p class="p2N"> The Round 5 of SELEX was performed by incubating for
+h15. The samples are then analyzed using a Nanodrop 2000
+          spectrophotometer. </p>
+        <table class="notTABLE">
+          <tbody>
+            <tr class="NotTR">
+              <td class="notTD">Sample</td>
+              <td class="notTD">Concentration (ng/µl)</td>
+              <td class="notTD">260/280 ratio</td>
+              <td class="notTD">260/230 ratio</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">SAr5</td>
+              <td class="notTD">30.3</td>
+              <td class="notTD">1.70</td>
+              <td class="notTD">1.03</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">EFr5</td>
+              <td class="notTD">33.7</td>
+              <td class="notTD">2.21</td>
+              <td class="notTD">1.74</td>
+            </tr>
+          </tbody>
+        </table>
+        <br>
+        <p class="p3N"> PCR 13 - Amplification of EFr5 and SAr5 </p>
+        <p class="p2N"> This PCR was performed to amplified the DNA after the
+          Round 5 of SELEX. The PCR followed the same program as described
+          previously with the same concentration. It was run overnight and hold
+          at 4°C. The samples are analyzed using a Nanodrop 2000
+          spectrophotometer. </p>
+        <table class="notTABLE">
+          <tbody>
+            <tr class="NotTR">
+              <td class="notTD">Sample</td>
+              <td class="notTD">Concentration (ng/µl)</td>
+              <td class="notTD">260/280 ratio</td>
+              <td class="notTD">260/230 ratio</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">PCR 13 - Round 5 - SA</td>
+              <td class="notTD">1228.2</td>
+              <td class="notTD">1.59</td>
+              <td class="notTD">0.99</td>
+            </tr>
+            <tr class="notTR">
+              <td class="notTD">PCR 13 - Round 5 - EF</td>
+              <td class="notTD">1334.8</td>
+              <td class="notTD">1.62</td>
+              <td class="notTD">1.02</td>
+            </tr>
+          </tbody>
+        </table>
+        <br>
+        </div>
+      </div>
+      <div id="PAGESE5" class="pages">
+        <div class="NCroix" id="croixSE5">
+          <div style="height:10%;width:80%;background-color:white;position:relative;top:-40%;transform-origin:50% 50%;transform:rotate(45deg);left:10%;border-radius:3px 3px;float:left;">
+          </div>
+          <div style="height:10%;width:80%;background-color:white;position:relative;top:-84%;transform-origin:50% 50%;transform:rotate(-45deg);left:10%;border-radius:3px 3px;float:left;">
          </div>
        </div>
@@ Line 3,933: / Line 4,442: @@
          </div>
          <div id="SE4" class="ent"  >
-            <p class="p1N"> July Week 2 &nbsp;: &nbsp;09.09.19 &nbsp;to
-           &nbsp; 09.13.19<br>
-        </p>
-        <p class="date">09.09.19</p>
-        <p class="p3N">Bacteria culture - <i>E. coli</i> containing the plasmid
-          PET28:GFP </p>
-        <p class="p2N">In order to extract the plasmid PET28:GFP, <i>E. coli</i>
-          was cultured on LB petri dishes containing kanamycin at 50 µg/mL. The
-          cultures were stored all night at 37°C. </p>
-        <p class="p3N">Bacteria culture - <i>E. faecium</i> and <i>S. aureus</i></p>
-        <p class="p2N"><i>E. faecium</i> and <i>S. aureus</i> were grown until
-          the OD reached between 0.5 and 0.6.</p>
-        <p class="p3N"> Digestion - EFr2 &amp; SAr2</p>
-        <p class="p2N">PCR samples from the Round 2 of SELEX were digested using
-          lambda exonuclease. A mix was performed in order to scale-up this step
-          and get enough digested material. <br>
-        </p>
-        <p class="p2N" ,="" style="margin-left:50px">1. Prepare the mix as
-          follows</p>
-        <table class="notTABLE">
-          <tbody>
-            <tr class="NotTR">
-              <td class="notTD"><br>
-              </td>
-              <td class="notTD">50µL reaction</td>
-              <td class="notTD">Mix</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">DNA (PCR11)</td>
-              <td class="notTD">4 µg</td>
-              <td class="notTD">120 µg</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">Lambda exonuclease buffer (10X)</td>
-              <td class="notTD">5µL</td>
-              <td class="notTD">µL</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">Lambda exonuclease</td>
-              <td class="notTD">1µL</td>
-              <td class="notTD">30µL</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">Nuclease-free H²0</td>
-              <td class="notTD">QSP 50 µL</td>
-              <td class="notTD">QSP 1.5 mL</td>
-            </tr>
-          </tbody>
-        </table>
-        <br>
-        <p class="p2N" ,="" style="margin-left:50px">2. Incubate samples for 30
-          min at 37°C in a water-bath</p>
-        <p class="p2N" ,="" style="margin-left:50px">3. Stop the reaction by
-          adding EDTA to 10 mM</p>
-        <p class="p2N" ,="" style="margin-left:50px">4. Heat at 75°C for 10
-          minutes to inactivate the enzyme</p>
-        <p class="p2N">Samples concentrations were checked using a Nanodrop 2000
-          spectrophotometer.</p>
-        <table class="notTABLE">
-          <tbody>
-            <tr class="NotTR">
-              <td class="notTD">Sample</td>
-              <td class="notTD">Concentration (ng/µl)</td>
-              <td class="notTD">260/280 ratio</td>
-              <td class="notTD">260/230 ratio</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">Dig-Round 2 - SA</td>
-              <td class="notTD">32.6</td>
-              <td class="notTD">1.91</td>
-              <td class="notTD">0.27</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">Dig - Round 2- EF</td>
-              <td class="notTD">20</td>
-              <td class="notTD">1.92</td>
-              <td class="notTD">0.25</td>
-            </tr>
-          </tbody>
-        </table>
-        <br>
-        <p class="p3N">Phenol/chloroform extraction with Isopropanol - Digested
-          products EFr2 &amp; SAr2<!--^--> </p>
-        <p class="p2N">Once digested, samples were purified after digestion.
-          They were analysed with a Nanodrop 2000 spectrophotometer.</p>
-        <table class="notTABLE">
-          <tbody>
-            <tr class="NotTR">
-              <td class="notTD">Sample</td>
-              <td class="notTD">Concentration (ng/µl)</td>
-              <td class="notTD">260/280 ratio</td>
-              <td class="notTD">260/230 ratio</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">EFr2 sample 1</td>
-              <td class="notTD">46.7</td>
-              <td class="notTD">1.47</td>
-              <td class="notTD">1.49</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">EFr2 sample 2</td>
-              <td class="notTD">42.1</td>
-              <td class="notTD">1.48</td>
-              <td class="notTD">1.52</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">SAr2 sample 1</td>
-              <td class="notTD">29.1</td>
-              <td class="notTD">1.66</td>
-              <td class="notTD">1.63</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">SAr2 sample 2</td>
-              <td class="notTD">37.3</td>
-              <td class="notTD">1.52</td>
-              <td class="notTD">1.55</td>
-            </tr>
-          </tbody>
-        </table>
-        <br>
-        <p class="p3N">Interpretation</p>
-        <p class="p2N">A total quantity of 692 pmol was obtained for <i>E.
-            faecium</i> in the Round 2, and 516 pmol for <i>S. aureus</i></p>
-        <p class="p3N">SELEX Round 3</p>
-        <p class="p2N">The first round of SELEX was performed with 1h45 hours of
-          incubation. The samples are then analyzed using a Nanodrop 2000
-          spectrophotometer.</p>
-        <table class="notTABLE">
-          <tbody>
-            <tr class="NotTR">
-              <td class="notTD">Sample</td>
-              <td class="notTD">Concentration (ng/µl)</td>
-              <td class="notTD">260/280 ratio</td>
-              <td class="notTD">260/230 ratio</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">SAr3</td>
-              <td class="notTD">33.6</td>
-              <td class="notTD">2.19</td>
-              <td class="notTD">1.45</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">EFr3</td>
-              <td class="notTD">42.9</td>
-              <td class="notTD">2.3</td>
-              <td class="notTD">1.76</td>
-            </tr>
-          </tbody>
-        </table>
-        <p class="p3N">PCR 11 - Amplification of EFr3 and SAr3</p>
-        <p class="p2N"> This PCR was performed to amplified the DNA after the
-          Round 3 of SELEX. The PCR followed the same program as described
-          previously with the same concentration. It was run overnight and hold
-          at 4°C. The samples are analyzed using a Nanodrop 2000
-          spectrophotometer. </p>
-        <table class="notTABLE">
-          <tbody>
-            <tr class="NotTR">
-              <td class="notTD">Sample</td>
-              <td class="notTD">Concentration (ng/µl)</td>
-              <td class="notTD">260/280 ratio</td>
-              <td class="notTD">260/230 ratio</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">PCR 11 - Round 3 - SA</td>
-              <td class="notTD">1086.6</td>
-              <td class="notTD">1.67</td>
-              <td class="notTD">1.20</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">PCR 11 - Round 3 - EF</td>
-              <td class="notTD">1496</td>
-              <td class="notTD">1.72</td>
-              <td class="notTD">1.38</td>
-            </tr>
-          </tbody>
-        </table>
-        <br>
-        <p class="date">09.10.19</p>
-        <p class="p3N">Bacteria culture - <i>E. coli</i> containing the plasmid
-          PET28:GFP</p>
-        <p class="p2N"> After an overnight growth, a colony of bacteria from the
-.09 was added into an erlenmeyer containing LB media. Then, the
-          erlenmeyer was put at 37°C all night under agitation at 140 rpm. </p>
-        <p class="p3N">Bacteria culture - <i>E. faecium</i> and <i>S. aureus</i></p>
-        <p class="p2N"><i>E. faecium</i> and <i>S. aureus</i> were grown until
-          the OD reached between 0.5 and 0.6 </p>
-        <p class="p3N">Digestion - EFr3 &amp; SAr3</p>
-        <p class="p2N"> PCR samples from the Round 3 of SELEX were digested
-          using lambda exonuclease. A mix was performed in order to scale-up
-          this step and get enough digested material. Samples concentrations
-          were checked using a Nanodrop 2000 spectrophotometer. </p>
-        <table class="notTABLE">
-          <tbody>
-            <tr class="NotTR">
-              <td class="notTD">Sample</td>
-              <td class="notTD">Concentration (ng/µl)</td>
-              <td class="notTD">260/280 ratio</td>
-              <td class="notTD">260/230 ratio</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">Dig - Round 3 - SA</td>
-              <td class="notTD">36.8</td>
-              <td class="notTD">1.92</td>
-              <td class="notTD">0.27</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">Dig - Round 3 - EF</td>
-              <td class="notTD">45.3</td>
-              <td class="notTD">1.81</td>
-              <td class="notTD">0.23</td>
-            </tr>
-          </tbody>
-        </table>
-        <br>
-        <p class="p3N"> Phenol/chloroform extraction with Isopropanol and
-          Glycogen - Digested products EFr3 &amp; SAr3 </p>
-        <p class="p2N"> Once digested, samples were purified after digestion
-          using a phenol/chloroform extraction. Glycogen for a final 0.5 μg/μL
-          concentration was added in order to maximise the amount of DNA
-          recovered and avoid pipetting the pellet. They were analysed with a
-          Nanodrop 2000 spectrophotometer. </p>
-        <table class="notTABLE">
-          <tbody>
-            <tr class="NotTR">
-              <td class="notTD">Sample</td>
-              <td class="notTD">Concentration (ng/µl)</td>
-              <td class="notTD">260/280 ratio</td>
-              <td class="notTD">260/230 ratio</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">EFr3 sample 1</td>
-              <td class="notTD">32.1</td>
-              <td class="notTD">1.56</td>
-              <td class="notTD">1.86</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">EFr3 sample 2</td>
-              <td class="notTD">22.7</td>
-              <td class="notTD">1.48</td>
-              <td class="notTD">1.89</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">SAr3 sample 1</td>
-              <td class="notTD">27.1</td>
-              <td class="notTD">1.52</td>
-              <td class="notTD">1</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">SAr3 sample 2</td>
-              <td class="notTD">65.4</td>
-              <td class="notTD">1.50</td>
-              <td class="notTD">1.24</td>
-            </tr>
-          </tbody>
-        </table>
-        <p class="p3N">Interpretation</p>
-        <p class="p2N"> A total quantity of 856 pmol was obtained for <i>E.
-            faecium</i> in the Round 3, and 516 pmol for <i>S. aureus</i>.
-          Because the purity of SAr3 sample 1 was really low, we only used SAr3
-          sample 2 for the following steps. </p>
-        <p class="date"> 09.11.19 </p>
-        <p class="p3N">Midiprep - <i>E. coli</i> containing the plasmid
-          PET28:GFP </p>
-        <p class="p2N"> The liquid culture of <i>E. coli</i> from the 10.09 was
-          centrifuged 10 minutes at 3,500 x g and the supernatant was removed.
-          Then, we used a midiprep kit from Qiagen to extract the plasmid from <i>E.
-            coli</i>. For that, the pellet was resuspended with 4 mL of P1
-          containing RNAse. Then, 4 mL of P2 was added and we waited 5 minutes
-          at RT. After that, 4 mL of P3 was added and the solution was mixed.
-          The solution was loaded on a column and the filtrate was recovered.
-          After, 4 mL of QBT Qiagen lip was added on the column and then the
-          filtrate was loaded on the column. After filtration, the column was
-          washed twice with QC buffer. In the filtrate, 5 mL of QF buffer and
-.5 mL of isopropanol were added. The solution was centrifuged during
-minutes at 15000 x g. The supernatant was removed and the pellet
-          was washed with 1 mL of ethanol 70% and centrifuged again 30 minutes
-          at 15,000 x g. We removed all the supernatant and let the pellet dry.
-          Then, 10 µL of TE buffer was added and the sample was vortexed.
-          Finally, the sample was stored at -20°C. The sample was checked with a
-          Nanodrop 2000 spectrophotometer. </p>
-        <table class="notTABLE">
-          <tbody>
-            <tr class="NotTR">
-              <td class="notTD">Sample</td>
-              <td class="notTD">Concentration (ng/µl)</td>
-              <td class="notTD">260/280 ratio</td>
-              <td class="notTD">260/230 ratio</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">pET28:GFP</td>
-              <td class="notTD">212.6</td>
-              <td class="notTD">1.88</td>
-              <td class="notTD">2.24</td>
-            </tr>
-          </tbody>
-        </table>
-        <br>
-        <p class="date">09.12.19 </p>
-        <p class="p3N"> Bacteria culture - <i>E. faecium</i> and <i>S. aureus</i>
-        </p>
-        <p class="p2N"><i>E. faecium</i> and <i>S. aureus</i> were grown until
-          the OD reached between 0.5 and 0.6</p>
-        <p class="p3N">SELEX Round 4</p>
-        <p class="p2N"> The Round 4 of SELEX was performed by incubating for
-h30. The samples are then analyzed using a Nanodrop 2000
-          spectrophotometer. </p>
-        <table class="notTABLE">
-          <tbody>
-            <tr class="NotTR">
-              <td class="notTD">Sample</td>
-              <td class="notTD">Concentration (ng/µl)</td>
-              <td class="notTD">260/280 ratio</td>
-              <td class="notTD">260/230 ratio</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">SAr4</td>
-              <td class="notTD">37.1</td>
-              <td class="notTD">2.02</td>
-              <td class="notTD">1.40</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">EFr4</td>
-              <td class="notTD">21.2</td>
-              <td class="notTD">2.25</td>
-              <td class="notTD">1.55</td>
-            </tr>
-          </tbody>
-        </table>
-        <br>
-        <p class="p3N"> PCR 12 - Amplification of EFr4 and SAr4 </p>
-        <p class="p2N"> This PCR was performed to amplified the DNA after the
-          Round 4 of SELEX. The PCR followed the same program as described
-          previously with the same concentration. It was run overnight and hold
-          at 4°C. The samples are analyzed using a Nanodrop 2000
-          spectrophotometer. </p>
-        <br>
-        <table class="notTABLE">
-          <tbody>
-            <tr class="NotTR">
-              <td class="notTD">Sample</td>
-              <td class="notTD">Concentration (ng/µl)</td>
-              <td class="notTD">260/280 ratio</td>
-              <td class="notTD">260/230 ratio</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">PCR 12 - Round 4 - SA</td>
-              <td class="notTD">1373.9</td>
-              <td class="notTD">1.62</td>
-              <td class="notTD">1.05</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">PCR 12 - Round 4 - EF</td>
-              <td class="notTD">1314.2</td>
-              <td class="notTD">1.62</td>
-              <td class="notTD">1.06</td>
-            </tr>
-          </tbody>
-        </table>
-        <br>
-        <p class="date"> 09.13.19 </p>
-        <p class="p3N">Bacteria culture - <i>E. faecium</i> and <i>S. aureus</i></p>
-        <p class="p2N"> <i>E. faecium</i> and <i>S. aureus</i> were grown
-          until the OD reached between 0.5 and 0.6 </p>
-        <p class="p3N"> Digestion - EFr4 &amp; SAr4 </p>
-        <p class="p2N"> PCR samples from the Round 4 of SELEX were digested
-          using lambda exonuclease. A mix was performed in order to scale-up
-          this step and get enough digested material. Samples concentrations
-          were checked using a Nanodrop 2000 spectrophotometer. </p>
-        <br>
-        <table class="notTABLE">
-          <tbody>
-            <tr class="NotTR">
-              <td class="notTD">Sample</td>
-              <td class="notTD">Concentration (ng/µl)</td>
-              <td class="notTD">260/280 ratio</td>
-              <td class="notTD">260/230 ratio</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">Dig - Round 4 - SA sample 1</td>
-              <td class="notTD">59.7</td>
-              <td class="notTD">1.79</td>
-              <td class="notTD">0.27</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">Dig - Round 4 - SA sample 2</td>
-              <td class="notTD">59.2</td>
-              <td class="notTD">1.80</td>
-              <td class="notTD">0.25</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">Dig - Round 4 - EF sample 1</td>
-              <td class="notTD">57.4</td>
-              <td class="notTD">1.80</td>
-              <td class="notTD">0.24</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">Dig - Round 4 - EF sample 2</td>
-              <td class="notTD">58.8</td>
-              <td class="notTD">1.79</td>
-              <td class="notTD">0.24</td>
-            </tr>
-          </tbody>
-        </table>
-        <br>
-        <p class="p3N"> Phenol/chloroform extraction with Isopropanol and
-          Glycogen - Digested products EFr4 &amp; SAr4 </p>
-        <p class="p2N"> Once digested, samples were purified after digestion.
-          They were analysed with a Nanodrop 2000 spectrophotometer. </p>
-        <table class="notTABLE">
-          <tbody>
-            <tr class="NotTR">
-              <td class="notTD">Sample</td>
-              <td class="notTD">Concentration (ng/µl)</td>
-              <td class="notTD">260/280 ratio</td>
-              <td class="notTD">260/230 ratio</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">EFr4 sample 1</td>
-              <td class="notTD">49.6</td>
-              <td class="notTD">1.46</td>
-              <td class="notTD">1.17</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">EFr4 sample 2</td>
-              <td class="notTD">34.1</td>
-              <td class="notTD">1.51</td>
-              <td class="notTD">1.13</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">SAr4 sample 1</td>
-              <td class="notTD">39.6</td>
-              <td class="notTD">1.54</td>
-              <td class="notTD">1.15</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">SAr4 sample 2</td>
-              <td class="notTD">18.7</td>
-              <td class="notTD">1.54</td>
-              <td class="notTD">1.33</td>
-            </tr>
-          </tbody>
-        </table>
-        <br>
-        <p class="p3N"> Interpretation </p>
-        <p class="p2N"> A total quantity of 650 pmol was obtained for <i>E.
-            faecium</i> in the Round 4, and 684 pmol for <i>S. aureus</i>.</p>
-        <p class="p3N"> SELEX Round 5 </p>
-        <p class="p2N"> The Round 5 of SELEX was performed by incubating for
-h15. The samples are then analyzed using a Nanodrop 2000
-          spectrophotometer. </p>
-        <table class="notTABLE">
-          <tbody>
-            <tr class="NotTR">
-              <td class="notTD">Sample</td>
-              <td class="notTD">Concentration (ng/µl)</td>
-              <td class="notTD">260/280 ratio</td>
-              <td class="notTD">260/230 ratio</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">SAr5</td>
-              <td class="notTD">30.3</td>
-              <td class="notTD">1.70</td>
-              <td class="notTD">1.03</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">EFr5</td>
-              <td class="notTD">33.7</td>
-              <td class="notTD">2.21</td>
-              <td class="notTD">1.74</td>
-            </tr>
-          </tbody>
-        </table>
-        <br>
-        <p class="p3N"> PCR 13 - Amplification of EFr5 and SAr5 </p>
-        <p class="p2N"> This PCR was performed to amplified the DNA after the
-          Round 5 of SELEX. The PCR followed the same program as described
-          previously with the same concentration. It was run overnight and hold
-          at 4°C. The samples are analyzed using a Nanodrop 2000
-          spectrophotometer. </p>
-        <table class="notTABLE">
-          <tbody>
-            <tr class="NotTR">
-              <td class="notTD">Sample</td>
-              <td class="notTD">Concentration (ng/µl)</td>
-              <td class="notTD">260/280 ratio</td>
-              <td class="notTD">260/230 ratio</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">PCR 13 - Round 5 - SA</td>
-              <td class="notTD">1228.2</td>
-              <td class="notTD">1.59</td>
-              <td class="notTD">0.99</td>
-            </tr>
-            <tr class="notTR">
-              <td class="notTD">PCR 13 - Round 5 - EF</td>
-              <td class="notTD">1334.8</td>
-              <td class="notTD">1.62</td>
-              <td class="notTD">1.02</td>
-            </tr>
-          </tbody>
-        </table>
-        <br>
-        </div>
-      </div>
-      <div id="PAGESE5" class="pages">
-        <div class="NCroix" id="croixSE5">
-          <div style="height:10%;width:80%;background-color:white;position:relative;top:-40%;transform-origin:50% 50%;transform:rotate(45deg);left:10%;border-radius:3px 3px;float:left;">
-          </div>
-          <div style="height:10%;width:80%;background-color:white;position:relative;top:-84%;transform-origin:50% 50%;transform:rotate(-45deg);left:10%;border-radius:3px 3px;float:left;">
-          </div>
-        </div>
          <div id="SE5" class="ent"  >

	50µL reaction	Mix
DNA (PCR11)	4 µg	120 µg
Lambda exonuclease buffer (10X)	5µL	µL
Lambda exonuclease	1µL	30µL
Nuclease-free H²0	QSP 50 µL	QSP 1.5 mL

Sample	Concentration (ng/µl)	260/280 ratio	260/230 ratio
Dig-Round 2 - SA	32.6	1.91	0.27
Dig - Round 2- EF	20	1.92	0.25

Samples	Concentration (ng/µl)	260/280nm ratio	260/230nm ratio
DNA library	2217.65	1.89	1.56
Reverse primer	8810.90	1.95	1.58

		Volume needed (μL)
Components	Final concentration	Mix 1	Mix 2	Mix 3
Phusion HF buffer 5X	1X	50	50	50
25 mM dNTPs	0.20 mM	2	2	2
Forward primer	0.50 μM	9.75	9.75	9.75
Reverse primer	0.50 μM	10.10	10.10	10.10
25 mM Magnesium	2.5 mM	25.0	50.0	75.0
DNA library	10.0 ng	22.5	22.5	22.5
Taq DNA polymerase	0.10 U/μL	0.02	0.02	0.02
Phusion DNA polymerase	1 U/μL	0.50	0.50	0.50
Nuclease-free water	/	130.13	105.13	80.13
Total volume	/	250	250	250

A (54°C)	1.1	2.1	3.1
B	/	/	/
C (51.9°C)	1.2	2.2	3.2
D	/	/	/
E (47.6°C)	1.3	2.3	3.3
F	/	/	/
G (44.7°C)	1.4	2.4	3.4
G	/	/	/

Sample	Concentration (ng/μL)	260/280nm ratio	260/230nm ratio
1.1	465.90	1.39	0.66
1.2	501.7	1.42	0.69
1.3	480.4	1.43	0.68
1.4	469.9	1.43	0.65
2.10	498.30	1.40	0.68
2.20	498.70	1.43	0.70
2.30	532.90	1.42	0.71
2.40	495.20	1.42	0.69
3.10	497.80	1.43	0.70
3.20	512.20	1.43	0.70
3.30	505.50	1.42	0.70
3.40	527.20	1.41	0.70

1.	Sample 1.1 (54°C, 25 μL, Mg2+)
2.	Sample 1.2 (51.9°C, 25 μL, Mg2+)
3.	Sample 1.3 (47.6°C, 25 μL, Mg2+)
4.	Sample 1.4 (44.7°C, 25 μL, Mg2+)
5.	Sample 2.1 (54°C, 50 μL, Mg2+)
6.	Sample 2.2 (51.9°C, 50 μL, Mg2+)
7.	Sample 2.3 (47.6°C, 50 μL, Mg2+)
8.	Sample 2.4 (44.7°C, 50 μL, Mg2+)
9.	Sample 3.1 (54°C, 75 μL, Mg2+)
10.	Sample 3.2 (51.9°C, 75 μL, Mg2+)
11.	Sample 3.3 (47.6°C, 75 μL, Mg2+)
12.	Sample 3.4 (44.7°C, 75 μL, Mg2+)

Components	Volume for 50 μL reaction
DNA from PCR 1	5 μg
Lambda Exonuclease Reaction Buffer (10X)	5 μL (1X)
Lambda Exonuclease	1 μL (5 units)
Nuclease-free water	up to 50μL

	Volume needed (in μL)
Components	Mix 1	Mix 2
Phusion HF buffer 5X	500	500
25 mM dNTPs	20	250
Forward primer	97.5	50
Reverse primer	101	50
25 mM Magnesium	250	250
DNA library (1/1000 dilution)	225	250
Taq DNA polymerase diluted 1/10	5	5
Phusion DNA polymerase	25	25
Nuclease-free water	1276.5	1135
Total volume	2500	2500

Sample	Pool 1 Elution 1	Pool 1 Elution 2	Pool 2 Elution 1	Pool 2 Elution 2
Upper aqueous phase recovered	2000 µL	430 µL	1150 µL	415 µL
Sodium acetate 3M to add	200 µL	43 µL	115 µL	41.5 µL
Volume after addition of sodium acetate	2200 µL	473 µL	1265 µL	456.5 µL
Ethanol 100% to add	5500 µL	1182.5 µL	3162.5 µL	1141.25 µL
Total volume	7700 µL	1655.5 µL	4427.5 µL	1597.75 µL

	Volume needed (in μL)
Components	Mix 1 (With DMSO)	Mix 2 (Without DMSO)
Phusion HF buffer 5X	50	50
25 mM dNTPs	5	5
Forward primer	10	10
Reverse primer	10	10
DMSO	7.5	0
Template DNA	22.5	22.5
Phusion DNA polymerase	2.5	2.5
Nuclease-free water	142.5	150
Total volume	250	250

Wells	Purification method	Concentration (ng/μL)	260/280 ratio	260/230 ratio
4	PCR product with DMSO	1157.6	1.64	0.85
5	PCR product without DMSO	1143	1.65	1.18
6	PCR clean up kit from QIAGEN with DMSO	7.9	2.26	-0.32
7	PCR clean up kit from QIAGEN without DMSO	15	2.07	-0.62
8	GE Healthcare columns with DMSO	34.5	0.96	0.34
9	GE Healthcare columns without DMSO	45.9	1.16	0.08
10	Gel extraction kit with DMSO	6.1	-6.66	0.01
11	Gel extraction kit without DMSO	-0.5	0.53	0

	Volume needed (in μL)
Components	Mix 1(10ng)	Mix 2(5ng)	Mix 3(1ng)
Phusion Buffer 10X	100	100	100
dNTPs 25 mM	10	10	10
Forward primer (1/10)	20	20	20
Reverse primer (1/10)	20	20	20
Template DNA (1/1000)	45	22.5	5
Phusion DNA polymerase	5	5	5
Nuclease-free water	300	322.5	340
Total volume	500	500	500

Sample	Concentration(ng/µl	260/280 ratio	260/230 ratio
PCR 8 pooled	1093.9	1.63	1.11

	S aureus
Components	SA 1 (1/10)	SA 2 (1/90)
Phusion buffer	10	10
dNTPs	1	1
Forward primer	2	2
Reverse primer	2	2
Template DNA (1/10)	1	0
Template DNA (1/90)	0	1
Phusion DNA polymerase	0.5	0.5
Nulease-free water	33.5	33.5
Total volume	50	50

	E. faecium
Components	SA 1 (1/10)	SA 2 (1/80)
Phusion buffer	10	10
dNTPs	1	1
Forward primer	2	2
Reverse primer	2	2
Template DNA (1/10)	1	0
Template DNA (1/80)	0	1
Phusion DNA polymerase	0.5	0.5
Nulease-free water	33.5	33.5
Total volume	50	50

Sample	Concetration (ng/μL)	260/280 ratio	260/230 ratio
EFr1 sample 1	17.2	1.46	1.29
EFr1 sample 2	26.5	1.49	1.44
EFr1 sample 3	29.8	1.49	1.47
EFr1 sample 4	37.2	1.56	1.5
SAr1 sample 1	69	1.51	1.54
SAr1 sample 2	61.5	1.75	0.28

Components	SAr1	EFr1
Phusion buffer	200	200
dNTPs	20	20
Forward primer	40	40
Reverse primer	40	40
Template DNA (1/3)	20	20
Phusion DNA polymerase	10	10
Nuclease-free water	670	670
Total volume	1000	1000