Standard PCR Conditions for Herman Lab (not for Expand PCR)

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Standard PCR Conditions for Herman Lab (not for Expand PCR)

Component Suggested range We use for 50 l rxn:
__ ul of __ stock
primers 20-500 pM 50 pM 2.5 of 20pM/l
buffer 1X 1X 5 of 10X
dNTPs 20-500 M 200 M 5 of 2 mM
MgCl2 1-9 M 2.5 M* 5 of 25 M
Taq 1-2.5 units 2 units 0.5 l
DNA template 105-106 (units?) ~50 ng genomic
~10 ng plasmid		 N/A
* unless it's in the buffer or you are varying the [Mg2+]

Master Mix I Master Mix II
annealing 5°C below lowest Tm of primer or close to that, no lower than 40°C
extension Temp: 72°C, Time: 1 min/Kb of expected product, 10 min on last cycle
denaturation Temp: 95°C, Time: 5 min on initial cycle and 30 sec to 1 min on rest
number of cycles 25-30, usually 30

Notes:
  • Primer stocks are 200 pmoles/l in TE pH 8.0
  • Use sterile ddH2O for dilutions and reactions
  • Filter tips should be used when getting into stocks (dNTPs, primers)
  • Make 100 l dilutions of primers in 0.6 ml tube

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