Lidstrom:PCR

Back to Protocols, Lidstrom

General Guidelines

• Design primers such that T_m is:
  • The T_m of each primer is between 55-65 °C (62 - 65 is best for Phusion)
    • If using Phusion, use the Finnzymes T_m calculator
  • The T_m of both primers are very similar, i.e., within ~2 °C
  • Either primer will not form a stable internal hairpin structure, i.e., ΔG <-3 kcal/mol
  • The forward and reverse primers do not combine to form a stable hairpin structure or dimer (check here)
  • If possible the 3' end of each primer should end with a GC
• Use the extension temperature stated on the kit -- being even 2° to high or too low is detrimental!
• Try T_annealing = 55°C first, then try other conditions if that fails.
• If a PCR isn't working:
  • there are more variables (annealing temperature, annealing time, concentration of several reagents, reaction additives) than you can play with, especially because they all have combinatorial effects on the result. When Janet gets stuck, she does what Justin Siegel proposed: 55°C, 60°C, 65°C, and 70°C ish and do each of these temperatures with 0%, 5%, and 10% DMSO. That makes 12 rxns; do on a gradient PCR block.
  • Also consider including positive and negative controls in repeat attempts.

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 Siegel)
• Amanda heard from a sales rep that the Finnzymes version is better than NEB's. I couldn't find supporting information and think this is a myth; finnzyme licensed the technology to NEB. (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. Janet 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.
  • You can also buy 2X mix that is a hot-start version of the enzyme, allowing you to set up your reactions at room temp. You pay even more for this.