Sean Lauber:PCR: Difference between revisions
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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 |
Latest revision as of 06:25, 19 July 2013
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