# Sean Lauber:PCR

(Difference between revisions)
 Revision as of 09:24, 19 July 2013 (view source)← Previous diff Current revision (09:25, 19 July 2013) (view source) Line 34: Line 34: For Kras genotyping (primers oIMR8274b, oIMR8273b and oIMR8272b) use x = 55*C For Kras genotyping (primers oIMR8274b, oIMR8273b and oIMR8272b) use x = 55*C + For Kras sequencing (primers SL038F and SL038R) use x = 55*C For Kras sequencing (primers SL038F and SL038R) use x = 55*C + For Cre recombination (primers CreRecF1 and CreRecR1) use x=60*C For Cre recombination (primers CreRecF1 and CreRecR1) use x=60*C

## Current revision

Note that this protocol and the reagent concentrations are meant for Life's platinum taq. If you are not using this Taq, make sure you change the concentrations.

For each reaction:

``` 18.15 μL  nuclease free water
2.5 μL    10X PCR buffer
0.5 μL    10 μM dNTPs
0.75 μL   50 mM MgCl2
1 μL      25 μM primer A
1 μL      25 μM primer B
1 μL      template (about 100 ng)
0.1 μL    Taq (platinum taq is good!)
------
25 μL     Total
```

Place in a thermocycler and program it as follows:

``` 94°C     3 min
```
``` 94°C     0.5 min|
x°C      0.5 min|  x35
72°C     1 min  |
```
``` 72°C     1 min
```

x: annealing temperature, this varies (search for annealing temp calculator to estimate from primer sequence)

### Common annealing temperatures for Richards lab

For Kras genotyping (primers oIMR8274b, oIMR8273b and oIMR8272b) use x = 55*C

For Kras sequencing (primers SL038F and SL038R) use x = 55*C

For Cre recombination (primers CreRecF1 and CreRecR1) use x=60*C