Team:SDU CHINA/Experiments
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Chemical Transformation
Materials:
LB broth
Ice
Selection plates
Methods:
1、The TOP10 receptor cells were taken out of -80'C, quickly inserted into the ice, and after 5 minutes, the bacteria were melted. The target DNA(plasmid or junction product) was added and mixed gently with the bottom of the EP tube by hand (avoid shooting with a gun). The cells were left in the ice for 25 minutes.
2、Heat shock at exactly 42°C for exactly 45 seconds. Do not mix.
3、Place on ice for 2 minutes. Do not mix.
4、Pipette 700 µl of room temperature 2YT or LB media into the mixture.
5、Incubate at 37°C and 200 rpm for 60 minutes.
6、Mix the cells thoroughly by flicking the tube and inverting.
7、Centrifuge at 5000 RPM for 1 minute to collect bacteria, and then take 100 microns of supernatant to gently blow the suspended bacteria and smear it on the corresponding antibiotic containing 2YT or LB medium.
8、Place the plate upside down in an incubator at 37℃ overnight for culture.
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Isolation of single colony - plate marking method
Materials:
LB Agar
Selection plates
pipette tips
Methods:
1、Divide the back of the petri dish into 12 areas and label them.
2、Using white tips selecting a bacterial colony and draw a curve on the sections of the selection plates.
3、Place the plate upside down in an incubator at 37℃ overnight for culture.
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Growing Overnight Cultures
Materials:
5 ml LB broth
5 μl antibiotic
12 ml culture tube
Methods:
Overnight cultures were prepared under sterile conditions using a Bunsen burner
1、Add 5 ml liquid LB media into 12 ml culture tubes
2、Add 5 μl of appropriate antibiotic into the broth
3、Using the pipette tip, pick a single colony and inoculate the cultures by dipping the tip into the LB broth
4、Seal the tubes and incubate overnight at 37°C shaking at 200-250 rpm
Note.
1、Culture tube carry bacteria:Add 5μl bacterial culture suspension into new selection culture tube,shaken overnight at 37℃.
2、Preservation strain restoration culture:Add 5μl bacterial and glycerol mix into new selection culture tube,shaken overnight at 37℃.
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PCR From Plasmid DNA Template
Materials:
ddHO
2 x Phanta Max Buffer
dNTP Mix (10 mM each)
10 µM forward primer
10 µM Reverse Primer
Phanta Max Super-Fidelity DNA Polymerase
PCR tube
Sterile water
Template DNA
Methods:
Normal PCR
For a 50 µL reaction
1、In a PCR tube on ice, combine the substances in the following table.Gently mix the reaction
reagent 50μl reaction system final concentration 2xEs Taq MasterMix (Dye) 25μl 1x Reverse Primer, 10 μM 2μl 0.4μM Template DNA <0.5μg <0.5μg/50μl ddHO Up to 50μl 2、Transfer the PCR tube from ice to a PCR machine preheated to 95°C to begin thermocycling.
Thermocycling
The PCR machine should be set to run the following steps:
Step Temperature (°C) Time Initial denaturation 94 30 seconds2 minutes denaturation 94 30 seconds annealing 55-65 25-35 cycles,30 seconds extension 72 30 seconds Final extension 72 2 minutes Note:
1、in general,the annealing temperature is 5°C lower than the melting temperature Tm of the amplified primer in the experiment.When the ideal amplification efficiency cannot be obtained,the annealing temperature should be appropriately lowered to increase the annealing temperature when the non-specific reaction occurs,so as to optimize the reaction conditions.
2、the extension time should be set according to the size of the amplified fragments.The amplification efficiency of Es Taq DNA Polymerase was 2 KB/min.
3、cycle number can be set according to the downstream application of amplification products.If the number of cycles is too small,the expansion increment is insufficient:if the number of cycles is too large,the mismatching probability will increase,and the non-specific background is serious.Therefore,the cycle times should be reduced as far as possible on the premise of guaranteeing the product yield.
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Preparation of LB Broth, Agar, and Glycerol Stocks
LB Broth:
Materials:
ddHO
5 g yeast powder
10g peptone
10g NaCl
1 Litre Purified Water
Methods:
1、Add 5 g yeast powder,10g peptone,10g NaCl to 1 litre purified water
2、High pressure steam sterilization at 121℃ for 20 minutes
LB Agar
Materials:
ddHO
5 g yeast powder
10g peptone
10g NaCl
20g agar powder
1 Litre Purified Water
Methods:
1、Add 5 g yeast powder,10g peptone,10g NaCl,20g agar powder to 1 litre purified water
2、High pressure steam sterilization at 121℃ for 20 minutes
Glycerol Stocks
Materials:
500µl glycerol (80%)
500µl overnight culture in LB
Methods:
1、Add 500µl glycerol (80%) to 1.5ml eppendorf tube
2、Add 500µl overnight culture in LB
3、Store at -80°C
M9 medium
Materials:
0.5g NaCl
3g KH2PO4
1g NH4Cl
30g Glucose
2ml 1M Mg2SO4 /p>
1ml 0.1M CaCl2
1 Litre Purified Water /p>
Methods:
1、Add material to 1 litre purified water
2、High pressure steam sterilization at 115℃ for 30 minutes
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DNA Gel Electrophoresis
Materials:
0.5xTBE buffer(45mM Tris,45mM boric acid,1mM EDTA,pH8.0)
1x TAE buffer(45mM Tris,45mM acetic acid ,1mM EDTA,pH8.0)
Gel mould
Gel Tank
8-10 µL DNA ladder
DNA loading dye(TBE/TAE)
10x loading buffer
DNA Marker
Methods:
1、Prepare 1% w/v solution of agarose powder in 0.5x TAE or 1x TAE buffer (e.g. 0.2g agarose powder in 20 mL buffer) using a conical flask
2、Heat the mixture until agarose is completely dissolved. Let the solution boil 3times.
3、Cooling and add 2 µL TBE/TAE dye into the solution.
4、Pour the solution into a gel mould. Make sure there are no bubbles in the solution.
5、Allows the solution to set (approx 15-20 minutes)
6、Transfer the agarose gel to a tank, remove the comb and apply.
7、Run the gel for 20-30 minutes at 120-130V
8、The position of the band was observed by uv irradiation at 365nm.
Note.TBE gel electrophoresis are used to detection,TAE gel electrophoresis are used to recycle.
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Adhesive recovery
1、After 20 minutes of operation at 120 V, the strip distribution was observed under 365 nm long wave ultraviolet. Use the clean blade to cut the target strip (try to remove gel without DNA, get the smaller volume of the gel), put it into 1.5ml EP pipe, and weigh the net weight of plastic block on the electronic scale.
2、Add equal volume PN solution according to the weight of rubber block, 0.1g rubber block corresponds to 100 μl PN solution. Melt in a metal bath at 50 ℃ and turn the EP tube gently to make it melt evenly.
3、In the adsorption column, 500 μl buffer BL was added and centrifuged at 12000rpm for 1min, and the column was in equilibrium.
4、The melted colloid was collected by centrifugation. After it was restored to room temperature, the solution was transferred into the adsorption column (the maximum capacity of the column was 800 μ l), and centrifuged at 12 000 rpm for 1 minute after standing for 2 minutes.
5、Remove the liquid from the adsorption column, add 600μl PW solution to it, and centrifuge at 12000rpm for 1min.
6、Remove the liquid from the column and repeat the above steps.
7、Remove the liquid in the adsorption column, centrifugate at 12000rpm for 2min.
8、Dry it on a 65 ℃ metal bath for 5min to remove ethanol, place the adsorption column on a clean 1.5ml ep tube, add 40μl water to the center of the membrane for elution, and centrifugate it at 12000rpm for 2min.
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Competent Cell Preparation
Materials:
1、SOB solution:
0.5% yeast extract
2% tryptone
10mM NaCl(0.581g/L)
2.5mM KCl(0.191g/L)
10mM MgCl2
10mM MgSO4
Dissolve in nanopure water and autoclave to sterilize.
2、TB solution:
10mM PIPES
15mM CaCl2(1.66g/L)
250mM KCl(18.6g/L)
Dissolve in nanopure water and adjust pH to 6.7 with KOH or HCl and then add MnCl2 to 55mM (10.88g/L), and adjust to final volume. Sterilize by filtration with 0.45um filter and store at 4°C
Methods:
1、Culture cells (DH5a in my case) on LB agar plate at 37°Covernight.
2、Pick up 10 -12 large colonies and culture in 250ml SOB in a 1L flask, 19°C with vigorous shaking to OD600=0.5 (normally it takes 24-36hrs)
3、Place the flask in ice for 10 min.
4、Pelleting the cell by spining at 4000rpm for 10 min at 4°C.
5、Gently resuspend the cell in 80ml ice-cold TB and store on ice for 10 min.
6、Spin at 4000rpm for 10 min at 4°C .
7、Gently resuspend the pellet in 20ml ice-cold TB and 1.4ml DMSO (the DMSO needs to be stored at -20°C o/n before use).
8、Aliquote the cell to 50 to 500ul for transformation or store at -70°C.
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Strain preservation
Take 1.5ml overnight culture solution, centrifugate at 12000rpm for 10min, remove the supernatant, add 300-500μl 15% glycerin, mix well, seal and label with sealant, and store at - 80 ℃.
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Demethylase cleavage
Each 50μl sample was added to 1μl DpnI treatment, recovery of agarose gel electrophoresis after 1 hour water bath at 37 ℃.
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Plasmid Extraction
1、Add 500μl balance liquid BL to the adsorption column CP3, centrifugate at 12000rpm for 1min, pour out the waste liquid in the collection pipe, and put the adsorption column back into the recovery header.
2、Take 1-5ml of overnight cultured bacterial solution and add it to the centrifuge tube, centrifuge at 12000rpm for 1min, and try to absorb the supernatant.
3、Add 250μl solution P1 to the centrifuge tube with bacterial precipitation, and use the pipette to completely suspend the bacterial precipitation.
4、Add 250μl solution P2 to the centrifuge tube, turn it up and down gently for 6-8 times to fully split the bacteria.
5、Add 350μl solution P3 to the centrifuge tube, turn it up and down gently for 6-8 times to fully split the bacteria.At this time, white precipitate will appear and centrifuged at 12000rpm for 10min.
6、Transfer the supernatant collected in the previous step to the adsorption column CP3 with a pipette, and do not suck out the precipitate. Centrifuge at 12000rpm for 1min, pour out the waste liquid in the collection pipe, and put the adsorption column into the collection pipe
7、Add 600μl PW solution to it, and centrifuge at 12000rpm for 1min.
8、Remove the liquid from the column and repeat the above steps.
9、Remove the liquid in the adsorption column, centrifugate at 12000rpm for 2min.
10、Dry it on a 65 ℃ metal bath for 5min to remove ethanol, place the adsorption column on a clean 1.5ml ep tube, add 80μl water to the center of the membrane for elution, and centrifugate it at 12000rpm for 2min.
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Overlap
ddHO 200 μl PrimeSTAR 200 μl forward primer(10 μM) 8μl Reverse Primerr(10 μM) <8μl First Template DNA 4μl Second Template DNA 4 μl
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PCR recycling
1、The PCR products were recovered into 1.5ml EP tube and 6-fold CP buffer was added, low speed centrifugation after mixing.
2、Put the HDM adsorption column into a 2ml collecting tube, add the above mixture into the HDM adsorption column, and centrifugate at 12000rpm for 1min.
3、Pour out the liquid in the collection tube, add 700μl wash buffer to the adsorption column, centrifugate at 12000rpm for 1min.
4、Pour out the liquid in the collection tube, repeat the previous step
5、Remove the liquid in the adsorption column, centrifugate at 12000rpm for 2min.
6、Dry it on a 65 ℃ metal bath for 5min to remove ethanol, place the adsorption column on a clean 1.5ml ep tube, add 40μl water to the center of the membrane for elution, and centrifugate it at 12000rpm for 2min.
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Growth characterisation
Materials:
Media of choice
Overnight culture of cells
12 well plate
Plate reader
Methods:
1、2ml system per hole
2、Blank group:2ml M9 medium
3、Control group:1.9ml M9 medium+0.1ml bacterial liquid+2μl antibiotic
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Gbison
Assembly master mixture 1251 μl 5× isothermal reaction buffer 500μl 10U/μl T5 exnuclease 1μl 2U/μl Phusion DNA polymerase 31.25 μl 40U/μl Taq DNA ligase 250 μl ddH2O 468.75 μl According to the concentration and length ratio of DNA fragments, 12μl system was evenly distributed: the short DNA fragments were added less, the high concentration DNA fragments were added less. Add the corresponding DNA fragment in proportion and mix it with the sub packed 12μl Gbison enzyme.
Metal bath or water bath 50 ℃ for 1 hour.
Put it in 4 ℃ refrigerator for preservation, and it must be transferred into the sensitive bacteria within one hour.
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12 hole plate
2ml system per hole:
Blank group:2ml M9 medium
Control group:1.9ml M9 medium+0.1ml bacterial liquid+1μl antibiotic