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Team:ESBS-Strasbourg/PCR
From 2008.igem.org
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
| Mother solution | Final concentration | Quantity, for 50 µl
of reaction mixture |
| 10X Taq buffer | 1X 5 µl | 5µL |
| 2 mM dNTP mix | 0.2 mM of each | 5µL |
| Primer I (10µM) | 0.1-1 µM | 2,5 µL |
| Primer II (10µM | 0.1-1 µM | 2,5 µL |
| Taq DNA Polymerase | 1.25 u / 50 µl | |
| 25 mM MgCl2 (we have it) | 1-4 mM | 5µL |
| Template DNA | 10ng/µL | |
| Water | _ | qsp |
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
