Difference between revisions of "Team:Pasteur Paris/Experiments"

Polymerase Chain Reaction

1) PCR Amplification using Takara Ex Taq DNA polymerase

• In a 0.2ml tube, set up the following reaction: tableau
• Set up the following cycles in a PCR machine
  • Initial denaturation : 94°C for 30 sec
  • 30 cycles :
  • 94°C for 30s
  • 55°C - 65°C for 1 min depending on your annealing temperature
  • 72°C 0.5-1 min per kb
  • Final extension: 72°C for 5min.

2) PCR Amplification using Phusion DNA polymerase

• In a 0.2ml tube, set up the following reaction: tableau
• Set up the following cycles in a PCR machine
  • Initial denaturation : 98°C for 30 sec
  • 30 cycles :
  • 94°C for 5-10s
  • 45°C - 72°C for 10 to 30s depending on your annealing temperature
  • 72°C for 15-30s per kb
  • Final extension: 72°C for 5min.

3) PCR Amplification using Q5 High Fidelity Master Mix DNA polymerase

• In a 0.2ml tube, set up the following reaction: tableau
• Set up the following cycles in a PCR machine
  • Initial denaturation : 98°C for 30 sec
  • 30 cycles :
  • 94°C for 5-10s
  • 45°C - 72°C for 10 to 30s depending on your annealing temperature
  • 72°C for 20-30s per kb
  • Final extension: 72°C for 2min.

Enzymatic Digestion

1) Single restriction enzyme digestion

• In a MicroCentrifuge tube, set up the following reaction on ice: tableau
• Pipette up and down to homogenize the solution.
• Quick spin in a MicroCentrifuge (5s).
• Incubation at 37°C for 1h.
• Heat inactivation for 20min at 80°C.
Optional
• Add the phosphatase: Add 1 unit of Shrimp Alkaline Phosphatase for each pmol of phosphate end.
• Incubate for 30min at 37°C.
• Inactivate the phosphatase at 65°C for 15 min.

2) Double digestion

• In a MicroCentrifuge tube, set up the following reaction on ice: tableau
• Pipette up and down to homogenize the solution.
• Quick spin in a MicroCentrifuge (5s).
• Incubation at 37°C for 1h.
• Heat inactivation for 20min at 80°C.
Optional
• Add the phosphatase: Add 1 unit of Shrimp Alkaline Phosphatase for each pmol of phosphate end.
• Incubate for 30min at 37°C.
• Inactivate the phosphatase at 65°C for 15 min.

Ligation

• Thaw the T4 DNA Ligase Buffer and DNA at room temperature.
• Set up the following reaction in a microcentrifuge tube on ice : tableau
• Gently mix the reaction by pipetting up and down
• Quick spin in a MicroCentrifuge (5s).
• For cohesive ends, incubation at 16°C for 1h.
• Heat inactivation for 10min at 65°C.

MiniPrep

1) Bacterial Culture Growth

• Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2–5 ml LB medium containing the appropriate selective antibiotic.
• Incubate for 8 h at 37°C with vigorous shaking (~300 rpm).

2) Plasmid purification

• Dilute the starter culture 1/500 to 1/1000 into selective LB medium.
• Grow at 37°C for 12–16 h with vigorous shaking (approx. 300 rpm).
• Centrifuge at 6000 x g for 15 min at 4°C.
• Resuspend the bacterial pellet in 4 ml Buffer P1.
• Add 4 ml Buffer P2, mix thoroughly by vigorously inverting the sealed tube 4–6 times
• Incubate at room temperature (15–25°C) for 5 min.
• Add 4 ml of chilled Buffer P3, mix immediately and thoroughly by vigorously inverting 4–6 times.
• Incubate on ice for 15 min.
• Centrifuge at ≥20,000 x g for 30 min at 4°C. Remove supernatant containing plasmid DNA promptly.
• Centrifuge the supernatant again at ≥20,000 x g for 15 min at 4°C. Remove supernatant containing plasmid DNA promptly.
• Equilibrate a QIAGEN-tip 100 by applying 4 ml Buffer QBT, and allow the column to empty by gravity flow.
• Apply the supernatant from step 8 to the QIAGEN-tip and allow it to enter the resin by gravity flow.
• Wash the QIAGEN-tip with 2x10ml BufferQC.
• Elute DNA with 5ml BufferQF.
• Collect the eluate in a 15 ml or 50ml tube.
• Add 3.5 ml (0.7 volumes) room-temperature isopropanol to the eluted DNA.
• Mix and centrifuge immediately at ≥15,000 x g for 30 min at 4°C.
• Carefully decant the supernatant.
• Air-dry the pellet for 5–10 min, and redissolve the DNA in a suitable volume of TE 8.1 Buffer.

Stab Cultures

• Prepare and autoclave 0.7% LB agar (standard LB medium containing 7 g/liter agar).
• Cool the LB agar to below 50°C (when you can hold it comfortably) and add the appropriate antibiotic(s). While still liquid, add 1 ml agar to a 2 ml screw-cap vial under sterile conditions, then leave to solidify.
• Using a sterile straight wire, pick a single colony from a freshly grown plate and stab it deep down into the soft agar several times.
• Incubate the vial at 37°C for 8–12 h leaving the cap slightly loose.
• Seal the vial tightly and store in the dark, preferably at 4°C.

MidiPrep

1) Bacterial Culture Growth

• Pick a single colony from a freshly streaked selective plate and inoculate a culture of 1–5 ml LB medium containing the appropriate selective antibiotic.
• Incubate for 12–16 h at 37°C with vigorous shaking.
• Centrifugation at > 8000 rpm (6800 x g) in MicroCentrifuge for 3 min at room temperature (15–25°C).
• Remove all traces of supernatant by inverting the open centrifuge tube until all medium has been drained.

2) Plasmid purification

• Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a micro- centrifuge tube.
• Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times.
• Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times.
• Centrifuge for 10 min at 13,000 rpm in a MicroCentrifuge.
• Apply the supernatants to the QIAprep spin column by decanting or pipetting.
• Centrifuge for 30–60 s. Discard the flow-through.
• Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through.
• Add 0.75 ml Buffer PE and centrifuge for 30–60 s.
• Discard the flow-through, and centrifuge at full speed for an additional 1 min.
• Place the QIAprep column in a clean 1.5 ml microcentrifuge tube.
• Add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column.
• Let stand for 1 min.
• Centrifuge for 1 min.

Transformation in chemically competent E.coli DH5-⍺

• Thaw out on ice one tube of chemically competent E.coli DH5-α.
• Place 50µL of cells in a pre-chilled MicroCentrifuge tube.
• Refreeze any unused cells.
• Add 1-10ng of DNA to the cells and mix gently. Do not mix by pipetting up and down.
• Incubate on ice for 30 min.
• Heat shock the cells for 40s in a 42°C water bath.
• Place the tubes on ice for 3 minutes.
• Add 700µl of prewarmed (37°C) SOC medium (Invitrogen NO 15544-034)
• Incubate at 37°C for 40 min at 200 rpm.
• Spread 200µL of transformed cells on the appropriate medium.
• Incubate overnight at 37°C

Agarose Gel Electrophoresis

• Prepare the different dilutions of TAE Buffer (50X, 1X, 0.5X)
• Agarose gel preparation :
• Weigh the agarose (Invitrogen Ref 16500-500) depending on the size of your DNA.
• Dissolve it in the appropriate amount of TAE Buffer (1X).
• Heat the preparation in the microwave until the agarose is dissolved.
• Cool the preparation by letting cool water flow against the Erlenmeyer until there is no evaporation.
• Add 1 drop of BET (Eurobio Ref GEPBET02-AF) under the extraction hood. Mix gently.
• Pour the solution in the casting tray and remove any bubbles.
• Place the combs in the casting tray and let it rest until the gel is solid.
• Carefully remove the combs from the gel
• Place the agarose gel in the electrophoresis apparatus filled with TAE buffer (0,5X).
• Gel loading:
• Load 2µl of DNA ladder in the first and eventually the last well.
• Load each well with the appropriate amount of DNA.
• Close the electrophoresis unit and run the gel for 1 hour at 130V and 7mA.

All the following steps take place under sterile conditions and on ice

• Inoculate 500mL of L-broth with 1/100 volume of a fresh overnight E. coli BAP1 culture.
• Grow the cells at 37 °C at 300 rpm.
• Chill cells on ice for about 20 min.
• Transfer the cells to a cold centrifuge bottle and spin at 4000 x g for 15 minutes at 4 °C.
• Carefully pour off and discard the supernatant. It is better to sacrifice a few cells than to leave supernatant behind.
• Gently re-suspend the pellet in 500 ml of ice-cold glycerol (10%).
• Centrifuge at 4000 x g for 15 minutes at 4 °C. Carefully pour off and discard the supernatant.
• Re-suspend the pellet in 250 ml of ice-cold glycerol (10%).
• Centrifuge at 4000 x g for 15 minutes at 4 °C. Carefully pour off and discard the supernatant.
• Re-suspend the pellet in ~ 20 ml of ice-cold glycerol (10%).
• Transfer to a 30 ml sterile Oakridge tube.
• Centrifuge at 4000 x g for 15 minutes at 4 °C. Carefully pour off and discard the supernatant.
• Re-suspend the cell pellet in a final volume of 2 ml of ice-cold 10% glycerol. The cell concentration should be about 3 x 1010 cells/ml.

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