Team:NTHU Taiwan/Experiments

Experiments

Cloning

PCR amplification

Normal PCR/ Gradient PCR (find best annealing temperature)

PCR mixture (YCL lab):

YCL lab Phusion polymerase premix(dNTP 10mM, Phusion pol, ddH2O, 5X buffer) 47ul
DNA template 1ul
Primer forward 10uM 1ul
Primer reverse 10uM 1ul
  • DNA amount: 100ng
  • primer concentration: 10uM

PCR program:

95&degC Preheat
95&degC 5min
95&degC 30s
60&degC (gradient determined best temp.) 30s
72&degC 2-3min, go back to step 3 repeat for 40 cycles.
72&degC 10min
4&degC o/n

Design:

Control: J04450 from miniprep

PCR cleanup (with Geneaid Gel/PCR Kit (DFH100, DFH300))
  1. Transfer reaction product to a 1.5 ml microcentrifuge tube. Add 5 volumes of Gel/PCR Buffer to the sample then vortex.(*If the mixture has turned from yellow to purple, add 10 µl of 3M sodium acetate (pH5.0) and mix thoroughly.)
  2. Transfer the sample mixture to the DFH Column. Centrifuge at 14-16,000 x g for 30 seconds. Discard the flow-through.
  3. Add 600 µl of Wash Buffer (make sure absolute ethanol was added) into the DFH Column and let stand for 1 minute. Centrifuge at 14-16,000 x g for 30 seconds then discard the flow-through. Centrifuge again at 14-16,000 x g for 3 minutes to dry the column matrix.
  4. Transfer the dried DFH Column to a new 1.5 ml microcentrifuge tube. Add 20-50 µl of water into the CENTER of the column matrix. Let stand for at least 2 minutes to allow water to be completely absorbed. Centrifuge at 14-16,000 x g for 2 minutes at room temperature to elute the purified DNA.
  5. If the final concentration of purified DNA is too low (lower than 15ng/ul), we would put the elution buffer (in our protocol is ddH2O in 55°c) with purified DNA back to the column and stand for 10 minutes to elute the remained DNA.
Digestion (20ul reaction)
  1. Set up the following reaction in a microcentrifuge tube on ice. (20ul reaction)
    DNA (depends on concentration) 1ug
    Cut Smart buffer (10X) 2ul
    Enzyme 1 0.5ul
    Enzyme 2 0.5ul
    • Digest for 1 to 2 hours
    • Use NED double digest finder (https://nebcloner.neb.com/#!/redigest)to find the best condition for digestion. EcoRI-HF, SpeI-HF, PstI and XbaI-HF are often used enzyme in iGEM.
    • Concentration of restriction enzyme should be lower than 5% to avoid star activity.
  2. Heat inactivate at 65°C / 80°C for 20 minutes.
    Design：
    Control: J04450 (if success, there will be two bands, at about 2000bp and 1000bp)
Cleanup (Same protocol as above)

Same protocol as above

Gel electrophoresis (Before ligation to determine the amount of DNA)
  1. Making 1.3% agarose gel (small)
    TAE buffer (1X) 25ml
    Agarose 0.33g
  2. Loading sample
    DNA marker: 1ul of 6X (loading dye/ Novel juice) + 5 ul of DNA ladder
    DNA sample: 1.5ul of 6X (loading dye/ Novel juice) + 2 ul of DNA
  3. Start the electrophoresis 100V for 30 minutes.
Ligation
  • Method 1: Use Nanodrop to measure the concentration of DNA sample and determine the volume used to ligate.
  • Method 2: Use the result of gel electrophoresis (brightness of DNA sample to determine the volume used to ligate.)
  1. Set up the following reaction in a microcentrifuge tube on ice. (20ul reaction)
    T4 DNA ligase 1ul
    T4 DNA ligase buffer (10X) 2ul
    Insert sequence The volume left in tube
    Backbone The volume of insert sequence /3
    ddH2O To 20ul
  2. Incubate at 16°C overnight
  3. Heat inactivate at 65°C for 10 minutes

Design:

Control: the product prepared from digestion and gel extraction (psB1c3 backbone and the mrfp part.)

Transformation
  1. Thaw a tube of DH5α Competent E. coli cells on ice.
  2. Add 3 µl containing ligation product to the 30ul of the cells. (DNA cannot exceed 10% of the total volume of competent cells.)
    PS: We wondered that the low concentration of ligation product may be the reason failing our experiment, so one time we alternate the protocol. We did PCR clean up after ligation, and transform all the ligation product, but it is still unsuccessful.
  3. Place the mixture on ice for 30 minutes.
  4. Heat shock at exactly 42°C for exactly 45 seconds.
  5. Place on ice for 5 minutes.
  6. Pipette 900 µl of room temperature LB broth without antibiotic into the mixture. (for recovery)
  7. Place at 37°C for 60 minutes.
  8. Warm selection plates to 37°C.
  9. Centrifuge at 400g to remove the excess LB and resuspend the cells with 200ul of LB with antibiotic. Spread onto a selection plate and incubate for 16 hours at 37°C.

Design:

Control: control group in ligation

Liquid culture
  1. Pick single colony with a 20-200ul tip.
  2. Put the tip into the tube with 5ml LB broth with antibiotic.
  3. Place at 37°C for 60 minutes. Shake vigorously (250 rpm).
Miniprep (with Geneaid Miniprep kit)
  1. Harvesting
    Transfer 1.0-1.5 ml of cultured bacterial cells to a microcentrifuge tube. Centrifuge at 14-16,000 x g for 1 minute at room temperature to form a cell pellet then discard the supernatant completely.
  2. Resuspension
    Add 200 µl of PD1 Buffer (make sure RNase A was added) to the 1.5 ml microcentrifuge tube containing the cell pellet. Resuspend the cell pellet completely by vortex or pipette until all traces of the cell pellet have been dissolved.
  3. Cell Lysis
    Add 200 µl of PD2 Buffer to the resuspended sample then mix gently by inverting the tube 10 times. Close PD2 Buffer bottle immediately after use to avoid CO2 acidification. Do not vortex to avoid shearing the genomic DNA. Let stand at room temperature for at least 2 minutes to ensure the lysate is homogeneous. Do not exceed 5 minutes.
  4. Neutralization
    Add 300 µl of PD3 Buffer then mix immediately by inverting the tube 10 times. Do not vortex to avoid shearing the genomic DNA. Centrifuge at 14-16,000 x g for 3 minutes at room temperature. If using >5 ml of bacterial cells, centrifuge at 16-20,000 x g for 5-8 minutes. During centrifugation, place a PDH Column in a 2 ml Collection Tube.
  5. DNA Binding
    Transfer all of the supernatant to the PDH Column. Use a narrow pipette tip to ensure the supernatant is completely transferred without disrupting the white precipitate. Centrifuge at 14-16,000 x g for 30 seconds at room temperature then discard the flow-through. Place the PDH Column back in the 2 ml Collection Tube.
  6. Wash
    Add 600 µl of Wash Buffer (make sure absolute ethanol was added) into the PDH Column. Centrifuge at 14-16,000 x g for 30 seconds at room temperature. Discard the flow through then place the PDH Column back in the 2 ml Collection Tube. Centrifuge at 14-16,000 x g for 3 minutes at room temperature to dry the column matrix. Transfer the dried PDH Column to a new 1.5 ml microcentrifuge tube.
    Perform Wash Buffer steps twice for salt sensitive downstream applications.
  7. Elution
    Add 50 µl of water into the CENTER of the column matrix. Let stand for at least 2 minutes to allow Elution Buffer, TE or water to be completely absorbed. Centrifuge at 14-16,000 x g for 2 minutes at room temperature to elute the purified DNA.

backbone

Transformation from iGEM kit
Liquid Culture
Miniprep
Digestion
Gel electrophoresis
Gel Extraction (If backbone from iGEM kit is digested, for example J04450 = pSB1C3 + mrfp, gel extraction is needed)
  1. Gel Dissociation
    1. Cut the agarose gel slice containing DNA fragments.
    2. Add 500 µl of Gel/PCR Buffer to the sample then mix by vortex.
    3. Incubate at 55-60ºC for 10-15 minutes or until the gel slice is completely dissolved. (During incubation, invert the tube every 2-3 minutes.) *If the color of the mixture has turned from yellow to purple, add 10 µl of 3M Sodium Acetate (pH5.0) and mix thoroughly. This will adjust pH and the color will return to yellow.
    4. Cool the dissolved sample mixture to room temperature.
  2. DNA Binding
    1. Place a DFH Column in a 2 ml Collection Tube.
    2. Transfer the sample mixture to the DFH Column.
    3. Centrifuge at 14-16,000 x g for 30 seconds. Discard the flow-through then place the DFH Column back in the 2 ml Collection Tube.
  3. Wash
    1. Add 400 µl of W1 Buffer into the DFH Column. Centrifuge at 14-16,000 x g for 30 seconds then discard the flow-through. Place the DFH Column back in the 2 ml Collection Tube.

      2. *The following steps are same as the third step onwards of PCR cleanup.

Protocol for OD value and Fluorescence intensity Measurement

Preparation of 10mM Oleic acid medium in LB
  1. Prepare 0.5% ethanol solution to dissolve fatty acid.
  2. Transfer 1mL NaOH in to Eppendorf, and add 31.5ul Oleic acid in it. Gently inverse the Eppendorf until the solution is well mixed.
  3. Transfer the Oleic-Alcohol solution 1ml into 9ml LB.
  4. Leave 10-15 min in agitation.
OD value and Fluorescence intensity Measurement
  1. Plate 180 ul Oleic acid LB into the wells of the 96-well plate.
  2. Add 20 ul cell culture into each well.
  3. Incubate the cells at 37°C.
  4. Use plate reader to trace OD value and Fluorescence intensity in an hourly basis.

Western Blot

CELL LYSIS
  1. Before start, precooled the PBS and centrifuger at 4°C.
  2. Prepare the lysis buffer composed of:
    • 50mM Tris pH 7.4
    • 50mM NaCl
    • 1% SDS
  3. Centrifuge the liquid culture of bacteria at 1000g for 5min and dispose the supernatant.
  4. Wash the cells with precooled PBS and centrifuge at 1000g for 5min.
  5. Aspirate the PBS and repeat the previous step for twice.
  6. Remove the PBS and add lysis buffer, then wait for 30min.
  7. Centrifuge with 16000g for 10 minutes at 4°C.
  8. Collect the supernatant to new microcentrifuge tubes and stored the samples in -32°c refrigerator.
BCA PROTEIN ASSAY (BICINCHONINIC ACID PROTEIN ASSAY)
  1. Take out the protein extraction kit and 96-well plate (can be reused)
  2. Mix Reagent A and Reagent B in the ratio of 50:1 (The volume of the mixture depends on the amount of wells used.)
  3. Drops each 100 μl of the mixed reagent into the wells.
  4. Then, making the standard curves by different volume of BSA (Bovine Serum Albumin) with concentration of 2 μg/μl. The volume is as followed:
    0μl of BSA 0.5μl of BSA 1 μl of BSA 2 μl of BSA Samples…
  5. Drops the left wells with 1 μl of samples.
  6. Wrap with plastic wrap and then place it in oven at 37ºC for 30 minutes.
  7. Measure the absorbance of samples for 562nm light.
  8. According to the absorbance measured, dilute the sample, calculation needed
    • Minus the blank solution absorbance and make a standard curve.
    • By using the gradient and y-intercept of the curve to determine the concentration of protein in the samples.
    • Select the lowest concentration of protein samples as the baseline and calculate the volume of PBS needed to dilute the other samples.
    • Calculate the volume of dye and loading buffer to be added. (Loading buffer: dye = 5:1)
  9. Heat at 95ºC for 10 minutes.
  10. Centrifuge 13000rpm for 1 minute. If you continue the SDS-PAGE immediately, then you can proceed to step 9 and 10. If not, step 9 and 10 and be skipped and do it when you want to do SDS-PAGE.
SDS-PAGE (SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS)
  1. Set up the apparatus and make sure it is not leaking by adding ddH2O into it.
    The setup of apparatus is like this:
    Resolving gel 12% (lower gel)
    Solution Volume used (μl)
    ddH2O 2000
    1.5M Tris-base (pH 8.6) 5000
    10% SDS 100
    30% acrylamide 4000
    TEMED 5
    10% APS 75

    * ddH2O should be added first to prevent precipitation. TEMED must be added the last because it will cause the solution to condense.

    *There are many other concentration of acrylamide gel which is suitable for different size of protein, please refer to the notes paste on the cupboard when making the gel.

    Protein size(kDa) Gel percentage (%)
    4-40 20
    12-45 15
    10-70 12.5
    15-100 10
    25-200 8
  2. Wipe off the ddH2O and add the lower gel. (*Little tips: Isopropanol can be added to flatten the gel and polymerize the gel faster.)
  3. Steaking gel
    Solution Volume used (μl)
    ddH2O 2810
    1.5M Tris-base (pH 8.6) 350
    10% SDS 56.5
    30% acrylamide 650
    TEMED 4
    10% APS 37.5
  4. Set up the apparatus.
  5. Loading the marker and samples, the volume of samples loaded depend on your samples’ concentration.
  6. Electrophoresis (20-30mA for one piece of gel).
COOMASSIE BLUE STAINING
  1. Stain the gel with 5cc Coomassie blue for 10mins.
  2. Wash the gel with ddH2O for 5-7 times or overnight.

    3. (If Western Blot is needed, then proceed to the next step.)

TRANSFER
  1. Immerse the PVDF membrane in methanol while the blot paper in 10x transfer buffer.
  2. Transfer with 20V for 60 minutes. (We can modify the transfer speed by adjust the current to 30mA)
BLOCKING
  1. 1. Prepared blocking solution, that is 5% non-fat dry milk in PBST and rock for 1 hour.
    (PBST is that PBS with 0.2% Tween 20, Tween 20 should be diluted into 20% first before prepared PBST)
    (The membrane can be rock for overnight but make sure in the -4°c refrigerator.)
INCUBATION
  1. Cut the PVDF membrane into two pieces according to the location of protein interest.
  2. Next, add in primary antibody in the ratio of 1:10000 to milk and then shake it for 1-2 hours under room temperature. To prevent wastage, we usually reuse the primary antibody. (time can be longer than that to get better effect.)
  3. Wash with PBST by shaking it for 10 minutes. Repeat thrice.
  4. Add in secondary antibody in the ratio of 1:10000 to PBST, shake it for 1-2 hours.
IMAGING
  1. Wash away the unbounded secondary antibody with PBST for three times.
  2. Prepared two types of ECL and mixed in the ratio of 1:1.
SUPPLEMENTARY

How to prepare reagents.

  1. 10X transfer buffer
    Tris base 15.2 g
    Glycine 72.1 g
    ddH2O To 500 ml
  2. 10X running buffer
    Tris base 30.3 g
    Glycine 144.1 g
    SDS 10 g
    ddH2O To 1 L
  3. 1.5 M Tris pH 8.8
    Tris-free base 90.75 g dissolved in 400ml ddH2O
    HCl Titrate to pH 8.8 (about 40-50 ml)
    ddH2O To 500 ml
  4. 1.0M Tris pH 6.8
    Tris-free base 60.54 g dissolved in 400ml ddH2O
    HCl Titrate to pH 6.8
    ddH2O To 500 ml

Lipase Activity Assay

Material
  1. 4-Nitrophenyl decanoate (pND)
  2. Acetonitrile
  3. HEPES buffer
  4. CaCl2
Procedure
  1. Substrate pND was dissolved in acetonitrile with concentration of 100 mM
  2. Reaction mixture: 5 µM CaCl2, 100 mM HEPES (pH 9), 1.25 mM substrate (pND), and 0.02 mg of enzyme, total volume 1 ml
  3. The amount of p-nitro-phenol released during hydrolysis was determined with spectrophotometric at 405 nm

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