Sean Lauber:PCR
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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