Designing Primers for Microsatellites
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Primers are necessary for two portions of the protocol in "fishing" out microsatellites. First, primers are necessary for the amplification of the segment of the vector containing the microsatellites. In addition, primers function in determining the sequence of the microsatellites with their flanking regions.
In the first instance, primers are selected from the regions of the vector on either side of the DNA digest insert. These primers are usually universal for all cloning vectors and either the T3/T7 primers or the M13 primers will work. Our vector had been modified somewhat by Invitrogen and only the M13 primers worked.
To design primers for sequencing of microsatellites, the amplified segment of DNA from the vector should be known. This sequence is copied (without base numbers or spaces included) into the Primer 3 program. This program can be accessed at the Primer3 web site.
Primers should be constructed to include the following parameters, having 18-20 base pairs, having a 55% GC content, and having a similar annealing temperature. The primer request should be assigned a sequence identification (Seq. ID). The program will give a suggested primer sequence for both forward (left) primer and reverse (right) primer. Check the parameters shown for that pair and, if acceptable, print them out. To check them further for self annealing and primer dimer formation, download the program Amplify (free program).
Enter the sequence of the primers, select the primers and request it to run PCR. If they have a high percentage of PCR production and low percentage of self-annealing, order the primers without fluorescent tags and try them out to be sure before investing in the more-expensive tagged primers.
An example of the process can be followed by clicking on Primers.
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