Team:ESBS-Strasbourg/PCR

I: Classic PCR

a) Remarks

-Use of the Taq polymerase -Te=72°C -Tm should be comprised between 55°C and 65°C. There is some exception, but in any case the Tm should inferior to the Te -Tm of primers should be very similar (a difference of about 2 degrees is accepted) -Gently vortex and briefly centrifuge all solutions after thawing -Adapt the protocol function of the mother concentration we have -We have to calculate the different volumes for a final volume of 50µL

b) Protocol

With the pFusion polymerase: primers for(10µM)  2,5µL primers rev(10µM)  2,5µL DNA template     10ng/µL Buffer + MgSO4    5µL dNTP(2mM)  5µL pFusion   1µL qsp water 50µL

PCR conditions: See the conditions commonly used, usually its a programm which is already saved on the machine Of course, just adjust the annealing temperature ;-)

II: Site directed mutagenesis

a) Remarks

-Use of the Fusion polymerase, for higher fidelity/processitivity -Tm higher, about 78°C (see primers table) -If the mother solutions own a different concentrations than those described below, then adapt the corresponding volume for the mix -Precisions could be add after the firsts experiments (e.g: for the PCR conditions, depends of how much it works) -Gently vortex and briefly centrifuge all solutions after thawing

b) Protocol

If we start with this solutions

-5x Phusion HF Buffer

-Phusion DNA polymerse (2u/µl)

-Primer 1 (5 µM )

-Primer 2 (5 µM )

-dNTPs 10 mM dATP, 10 mM dCTP, 10 mM dGTP, 10 mM dTTP

Then we have this protocol:

Mix:

10 µl tampon enzyme 5x concentré

1 µl dNTPs à 10 mM (200 µM de chaque)

x µl de plasmide (1 pg-10 ng)

5 µl du primer 1 (5 pmol/µl) (0.5 µM final) 5 µl du primer 2 (5 pmol/µl) (0.5 µM final) 0,5 µl de Phusion DNA polymearse (0.02 u/µl final)

H20 (q.s.p. 50 µl)

PCR conditions:

Step 1 : denaturation 30 s at 98°C Step 2 : denaturation 10 s at 98°C Step 3 : hybridation 30 s at Tm-2°C Step 4 : elongation x (15-30 s/Kb) mn at 72°C Step 5 : back to step 2, x time Step 6 : elongation 5-10 mn at 72°C Step 7 : maintain at 4°C

Keep the resulting solution at -20°C until to use it

Protocols