Team:ESBS-Strasbourg/PCR

From 2008.igem.org

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'''I:Classic PCR''' <br>
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'''I: Classic PCR''' <br>
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----
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a)Remarks: <br>
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a) Remarks <br>
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-We have to calculate the different volumes for a final volume of 50µL
-We have to calculate the different volumes for a final volume of 50µL
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b)Protocol: <br>
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b) Protocol <br>
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|25 mM MgCl2 (we have it)    ||    1-4 mM || 5µL
|25 mM MgCl2 (we have it)    ||    1-4 mM || 5µL
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|Template DNA          ||    10pg-1µg ||
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|Template DNA          ||    10ng/µL ||
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|Water          ||  _ || qsp
|Water          ||  _ || qsp
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With the pFusion polymerase: <br>
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primers for(10µM)  2,5µL <br>
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primers rev(10µM)  2,5µL <br>
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DNA template      10ng/µL <br>
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Buffer + MgSO4    5µL <br>
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dNTP(2mM)  5µL <br>
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pFusion    1µL <br>
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qsp water 50µL <br>
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'''II:Site directed mutagenesis'''
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'''II: Site directed mutagenesis'''
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a)Remarks: <br>
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a) Remarks <br>
-Use of the Fusion polymerase, for higher fidelity/processitivity <br>
-Use of the Fusion polymerase, for higher fidelity/processitivity <br>
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-Gently vortex and briefly centrifuge all solutions after thawing <br>
-Gently vortex and briefly centrifuge all solutions after thawing <br>
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b)protocol: <br>
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b) Protocol <br>
If we start with this solutions <br>
If we start with this solutions <br>

Latest revision as of 12:44, 21 July 2008

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


Protocols