Lidstrom:PCR

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General Guidelines

• Use the extension temperature stated on the kit -- being even 2 degrees to high or too low is detrimental!
• If a PCR isn't working there are more variables than you can possibly change because they all have combinatorial effects on the result.
  • First consider including positive and negative controls in your repeat attempts before trying to change the rxn conditions.
  • Janet always does the rxn at 55oC first and if that fails, you can try using 55oC, 60oC, 65oC, and 70oC ish and do each of these temperatures with 0%, 5%, and 10% DMSO.

Taq

• Cheap
• Use for colony PCR (you don't keep the DNA so higher error rattes are ok)

Phusion

• faster, more accurate, & higher processiviy
• use for DNA modifications (e.g. things that will be put in vivo)
• more details & instructions: Phusion
• more stable (DNA/polymerase or tRNA/DNA?) interactions at high annealing temps (said Justin Siegal)
• Amanda heard that the Finnzymes version is better than NEB's. I couldn't find supporting information. (Janet)

General Notes

• When finishing a PCR cycle please cancel or stop all running programs before turning the thermocycler off. Turning certain thermocyclers off with a running program can cause machine errors or erratic behavior when turning the machine back on again. -Andrew Lamb 2/22/12

Buying Reagents

• Phusion
  • You can buy from NEB or finnzymes; Janet uses NEB. You can buy the pure enzyme or a 2X master mix solution wherein they added the nucleotides for you. You pay ~50% more per rxn when you use the 2X version put there are pros: (a) it does save some time (b) it prevents the possibility of forgetting to add dNTPs and (c) saves you the cost/hassle of buying a set of dNTPs separately. [User:Janet B. Matsen] I called customer support on 3/6/2012 to ask whether I can make my own 2X mix from the less expensive pure enzymes. They do add a stabilizer to the 2X version (probably for the nucleotides) and they can't tell me what it is. They plan to get back to me when they ask experts whether I can make my own 2X anyway.