Hi, I transform my gene of interest+ MYC into blunt end vector (my gene of interest+ MYC is sticky end with SpeI) so for doing colony PCR can I use blunt end vector Reverse & Forward primers instead of using forward primer of my gene of interest and reverse primer of MYC?If no so why whats reason?
If vector primers do not contain SpeI site, it is possible to use them for colony PCR.Using of vector primers can be more informative, because there always will be PCR products irrespectively of insertion presence.
However, one should be noted, that if your insertion is long, elongation time should be enough for proper amplification. When elongation is too short, it is possible do not get amplicon from plasmid with insertion. In such circumstances using of primers set, where one primer is complementary to vector, and one to gene, will be more advantageous, giving short DNA product.
My gene product is totally 972bp, but pEasy-Blunt Zero vector (3956bp) have SpeI, PstI , PmeI, NotI sites so I can not use vector primers. Is it right @Igor Oscorbin
According to vector map, M13 forward and reverse primers flank SpeI site. Insertion is short, so it is possible to use M13 primers for colony PCR in that case.
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