Design of PCR primers


To amplify a few kb of a known region (to check genome sequence data, to sequence various individuals, to find SNP,etc)

- the forward primer should be at least about 50 bp away from the beginning and the reverse primer and at least about 50 kb away from the end of the sequence of interest.
- select a region with many G/C which is between 18 and 22 nucleotide long.
- calculate the Tm of this putative primer using the following web page:
http://www.basic.northwestern.edu/biotools/oligocalc.html
- the Tm value given on the first line (°C (Basic)) should be between 52°C and 56°C. If you can't find such a high Tm/high G/C-rich region, then choose a Tm around 50°C.
- both primer pairs should have a Tm difference that is strictly lower than 2°C. The same Tm for both primers is best. To adjust Tm, change the size of one of your two primers (should always be between 18 and 22 nucleotide long).
- try to have A or T as the last 3' nucleotide for each of your primers.
- If your forward and reverse primers are more than 1.2 kb away from each other, then design several pairs of primers to amplify overlapping fragments covering your entire region of interest.

- perform the PCR reaction with the GoTaq mix using a hybridization temperature equals to (Tm - 2°C).
- if you get a single and strong band, send directly for purification and sequencing using one of your primer pair.
- if you get several bands, increase hybridization temperature until you get a single band.