Hi, I have recently produced a clone library featuring the microsatellite sequences from four different primer pairs. Following TA cloning I have performed four seperate PCRs upon the colonies with the different primer pairs in order to discover which fragment has been taken up by each colony. Some of the colonies appear to amplify with more than one primer pair, indicating the possibility of multiple inserts.
I have sequenced some of these colonies using both the recommended insert primers for the cloning vector and the individual microsatellite primer pairs. The results are rather confusing, with the sequences produced using the insert primers clearly indicating a single microsatellite fragment to be present, flanked on either side by the insert region of the cloning vector. However cloning of the same colonies using the primer pairs of some of the other microsatellites featured in the library has uncovered the sequences of some of these microsatellites. These sequences do not appear in the fragment sequenced using the insert primers, so I have no idea where they are from.
I do not believe that the microsatellite amplification is due to non-specific amplification of either the cloning vector or the transformed E. coli as the sequences are close to those of the target species (which is an invertebrate) and the annealing temperature of some of the primer pairs are around 60C.
I'm not totally clear on all of the data you have, but something to consider when doing PCR analysis of colonies. You can actually amplify non-plasmid DNA from a plate if enough spread on the plated. This happens if PCR product unincorporated into a plasmid is picked up when you pick you colony, and, due to the sensitivity of the PCR, is amplified giving a false positive, or in your case, double-positive.
You may get a more specific PCR screen by using a primer-pair that will only work when a complete plasmid is formed, such as one of your target-specific primers combined with a vector-specifc primer (T7, M13, BGH oer whatever generic priming site your vector may have). This MAY help select for complete plasmid, but won't help in the case of two plasmids in one colony.
I assume all of your amplicons are the same size? If not, you could try using the vector-specific primers and look for the product with the size-of-interest.
Edited by wirly, 09 November 2009 - 07:48 PM.