Cloning by PCR
Cloning by PCR
This is the basic PCR method to amplify your DNA from a plasmid using the Phusion polymerase. You also use this protocol for an EIPCR reaction.
The oligo concentrations in your stocks should be 100uM. You use them at 10uM in this protocol. So, you first need to make an oligo dilution of:
9uL Water 1uL 100uM oligo
You can throw away the remainder of the diluted oligo when you are done, but hold onto your stock tube!
Set up the following reaction in a PCR tube:
31.5uL ddH2O 10uL 5x Phusion Buffer 5uL dNTPs (2mM in each) 1uL Oligo 1, 10uM 1uL Oligo 2, 10uM 1uL Template DNA 0.5uL Phusion Polymerase
The Expand temperature programs are a little complicated, so I won’t write them out here. They all involve 2 distinct cycling phases—the first 10 cycles have shorter extensions than the latter 20 cycles. You choose which one you want based on the length of your predicted product. If it is under 2kb, use 2K55. If it is between 2kb and 4kb, use 4K55. If it is over 4kb, use 8K55. In each case, start with the "55" version of the program. If you get no product or poor yield of product, repeat the same PCR reaction as two different variants, both with the “45” program. The first reaction will be the same composition as the first. For the second one, don’t add 3.3 uL of the water and instead use the volume for 3.3 uL of DMSO (dimethylsulfoxide). If these PCRs fail as well, well you'd either redesign, use a different template source, or give up.