PCR amplification and applications

PCR AMPLIFICATION AND APPLICATIONS
	Last Update: December 2006


PROCEDURE - standard PCR
The polymerase chain reaction is such a basic tool that its uses are too vast to even try to list them here. Every application has its own specifics and the researcher should take into consideration every single aspect of this reaction in order to be successful. Reading the various books available on this topic is recommended
Primer design: highly dependent on the experiment. Programs are available for this purpose. Rule of thumb: add 4^oC for every G/C and 2^oC for every A/T when calculating the annealing temperature
1. Into a 0.5 mL microcentrifuge tube add: 1 mL of DNA (30 ng of DNA is more than enough), 5 mL of 10 X PCR buffer (the final concentration of MgCl₂ can be optimized between 0.5 to 5mM), 4 mL of 5mM dNTP mix, 1 mL of sense primer (10-20 pmoles/mL), 1 mL of antisense primer (10-20 pmoles/mL), 0.3 mL of Taq DNA polymerase (1-5 U) and dH₂O to 50 mL
2. Mix and spin down reaction
3. Run the reaction following these PCR conditions: 94^oC for 1-3 minuts (denature DNA, specially important when using genomic DNA), followed by 20 to 30 rounds of PCR amplification (steps 1 through 3): 1. 94^oC for 20-60 sec (denature DNA), 2. 50-65^oC for 30-90 sec (anneal primers to DNA), 3. 72^oC for 60-180 sec (elongation of amplified product). One last step to finish the amplificatino of fragment(s): 72^oC for 5-10 mins (depending on the size of the amplicon). Store at 4^oC (or -20^oC) until analyzed
4. Analyze by agarose electrophoreses
NOTE: polymerases like Pwo (Roche) are proofreading polymerases, which means that they have a much lower rate of errors in the amplified product. At the same time, their products lack any addition of nucleotides at the end of the amplified product (in the case of Taq polymerase, an adenine nucleotide tends to be added at the end of the amplified product). Because of this, Pwo products can be used directly for blunt cloning, as in the case of site directed mutagenesis.

PROCEDURE - Site directed mutagenesis
The same idea as in any kind of PCR, but in this case the primers are designed to amplify the whole vector (+insert) and contain the mutation that is desired. The forward primer and the reverse primer should be pointing away from each other, and their 3'-ends should end exaclty next to each other. Either one of these two primers should be phosphorylated (required for blunt ligation) or the amplified product would need to be phosphorylated before ligation. The insertion of the mutation can be done on either primer by simply introducing the desired modification(s)

PROCEDURE - Colony PCR
This protocol uses a small portion of a bacterial colony as the single source of DNA for a PCR reaction. This reduces the time needed to check clones since there is no need to grow the colony and perform a mini-prep to obtain the plasmid DNA
1. Pick a small portion of a colony of interest
2. Put it in 50 mL of dH₂O
3. Boil for 10 mins
4. Spin at 10,000 rpms for 2 mins
5. Use 10 mL of supernatant in a PCR reaction