Sean Lauber:PCR

From OpenWetWare
Jump to: navigation, search

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