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:

    •  

    Tube

    Control

    Control

    Control

    Control

    Ex taq Buffer (10X)

    5µl

    5µl

    5µl

    5µl

    5µl

    dNTP mix

    4µl

    4µl

    4µl

    4µl

    4µl

    Template DNA

    1-10ng

    1-10ng

    1-10ng

    1-10ng

     

    Forward Primer

    final concentration: 0.2µM

     

    final concentration: 0.2µM

     

    final concentration: 0.2µM

    Reverse Primer

    final concentration: 0.2µM

     

     

    final concentration: 0.2µM

    final concentration: 0.2µM

    Nuclease free water

    to 50µl

    to 50µl

    to 50µl

    to 50µl

    to 50µl

    Ex Taq DNA Polymerase

    0.25µl

    0.25µl

    0.25µl

    0.25µl

    0.25µl

    Total

    50µl

    50µl

    50µl

    50µl

    50µl
  • 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.

Preparation of Electro-competent E.coli BAP1.

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.

Electroporation of Electrocompetent E.Coli BAP1

  • Thaw the cells on ice.
  • In a cold, 1.5 ml polypropylene microfuge tube, mix 40μL of the cell suspension with 5μL of DNA (DNA should be in a low ionic strength buffer such as TE).
  • Mix well and incubate on ice for 1 minute.
  • Set the MicroPulser to “Ec1” : for 0.1 cm cuvettes U=1,8kV and 1 pulse.
  • Transfer the mixture of cells and DNA to a cold electroporation cuvette and tap the suspension to the bottom.
  • Place the cuvette in the chamber slide.
  • Push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber.
  • Pulse once.
  • Remove the cuvette from the chamber and add 2 ml of SOC medium to the cuvette.
  • Quickly but gently re-suspend the cells with a Pasteur pipette.
  • Transfer the cell suspension to a 17 x 100 mm polypropylene tube and incubate at 37 °C for 1 hour, shaking at 225 rpm.
  • Plate 200μL of the cell suspension on a Petri Dish LB+ appropriate antibiotic.
  • Incubate overnight at 37 °C.

QIAgen Gel Extraction Kit Protocol

  • Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
  • Weigh the gel slice in a colorless tube
  • Add 3 volumes Buffer QG to 1 volume gel (100 mg ~ 100 μl). For >2% agarose gels, add 6 volumes Buffer QG.
  • Incubate at 50°C for until the gel slice has completely dissolved. Vortex the tube every 2–3 min to help dissolve gel.
  • Check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 μl 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow.
  • Add 1 gel volume of isopropanol to the sample and mix.
  • Place a QIAquick spin column in a provided 2 ml collection tube.
  • Apply the sample to the QIAquick column and centrifuge for 1 min.
  • Discard flow-through and place the QIAquick column back into the same tube.
  • To wash, add 0.75 ml Buffer PE to QIAquick column and centrifuge for 1 min
  • Discard flow-through and place the QIAquick column back into the same tube.
  • Centrifuge the QIAquick column once more in the provided 2 ml collection tube for 1 min at 17,900 x g to remove residual wash buffer.
  • Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
  • To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the QIAquick membrane and centrifuge the column for 1 min.
  • Let the column stand for 1 min,
  • Centrifuge for 1 min.
  • If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.
  • image

pNP-Assay

  • Preparation of PCS Buffer (1M, pH=7.4)
    • 8g of NaCl
    • 0.2g of KCl
    • 1.44g of Na2HPO4
    • 0.24g of KH2PO4 
    • 1L distilled H2O.
  • Preparation of 4-Nitrophenylbutyrate (pNP) in H2O.
    • Add 88µL of pNP in 912µL acetonitrile (500mM in ACN)
    • Dilute to get 1ml of pNP solution at 1mM
  • Preparation of the Bacterial culture at OD(600nm)=0.1
    • Measure OD(600nm) of bacterial suspension.
    • Dilute the bacterial suspension with PBS buffer.
  • Tableau
  • In each well of a 96-wells plate (flat-bottom), add 100µL of bacterial suspension (OD(600nm)=0.1))
  • Incubate at 34°C.
  • In each well, add 10µl of pNP solution.
  • For 30min, OD(405nm) is recorded every minute for each solution.

Preparation of Electrocompetent Saccharomyces Cerevisiae cells.

  • Inoculate 500mL of YPD in a 2.8 L fembach flask with an aliquot from an overnight culture of < Saccharomyces Cerevisiae.
  • Incubate at 30°C overnight, shaking at 250 rmp.
  • Chill the cells on ice water for 15min to stop growth.
  • Decant the cells into to sterile 250 ml centrifuge bottles and pellet the cells by centrifugation at 3000 X g for 5min at 4°C.
  • Carefully pour off and discard the supernatant ; place the centrifuge bottles with the cell pellets on ice.
  • Add 50ml of sterile, ice-cold water to each of the bottles and vortex to resuspend the cell pellets 
  • Bring the volume in each of the centifuge bottles to 250ml.
  • Pellet the cells by centrifugation at 3000 X g for 5min at 4°C ; pour off and discard the supernatant.
  • Wash the cells with a total of 250mL sterile, ice cold water.
  • Resuspend the cell pellet in 20mL of sterile, ice cold Sorbitol(1M) and transfer to a chilled 30ml Oakridge tube.
  • Pellet the cells by centrifugation at 3000 X g for 5min at 4°C
  • Pour off and discard the supernatant.
  • Resuspend the cells pellet in 0.5 mL of sterile, ice cold sorbitol  ; the final cell volume should be around 1.3 mL.

Electroporation in Electrocompetent Saccharomyces Cerevisiae cells.

  • Pipette the DNA samples (5-100ng) to be electroporated into sterile 1.5 mL microfuge tubes. Place tubes on ice.
  • Add the competent cells:
    • If 0.2 cm cuvettes are used, add 40 uL of the competent cells to each DNA sample
    • If 0,4 cm cuvettes are used, add 80 uL of the competent cells to each DNA sample.
  • Mix gently and incubate on ice for 5min.
  • Set the MicroPulser to « Sc2 » when using 0.2 cm cuvettes or to « Sc4 » when using 0.4 cm cuvettes. See Section 4 for operating instructions.
  • Transfer the DNA-cell samples to the appropriate electroporation cevettes that have been chilled in ice and tap the suspension to the bottom of the tube.
  • Place the cuvette in the chamber slide.
  • Push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber.
  • Pulse once.
  • Remove the cuvette from the chamber and immediatly add 1mL of ice cold sorbitol (1M) to the cuvette. Gently transfer the diluted cells into a sterile tube.
  • Check and recold the pulse parameters. The time constant should be close to 5 milliseconds.
  • Plate aliquots of the electroporated cells on selective agar plates containing sorbitol (1M).

Transformation of SK1 Yeast by electroporation

  • Inoculate 10 ml of YPD with a yeast strain and grow to saturation at 30°C with shaking.
  • Dilute into 40 ml of YPD in a 250 ml or larger sterile flask and grow for 2 hr at 30°C with shaking.
  • Collect the cells by centrifugation at 1,000 X g for 5 min.
  • Decant the supernatant and resuspend the pellet in 18 ml of TE Buffer. Then add 2 ml of 1 M Lithium Acetate.
  • Incubate cells at 30°C on a roller drum for 45 min.
  • Add 500 μl of DTT (1M).
  • Incubate for 15 min at 30°C on a roller drum.
  • Add 80 ml of sterile ddH2O at room temperature.
  • Centrifuge cells at 1,000 X g for 5 min.
  • Decant the supernatant and resuspend the pellet in 100 ml of sterile ddH2O.
  • Again centrifuge cells at 1,000 X g for 5 min.
  • Decant the supernatant and resuspend the pellet in 5 ml of 1 M Sorbitol.
  • Centrifuge cells at 1,000 X g for 5 min.
  • Decant supernatant and put cells on ice. Resuspend the pellet in 120 μl of cold Sorbitol (1M). The volume of resuspended cells should be around 180 μl.
  • Keep cells on ice and mix in sterile microfuge tubes
    • 40 μl of competent yeast cells
    • 1.7 μl of Carrier DNA (15 mg/ml)
    • DNA to be transformed (up to 5 μl)

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