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

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