I cloned a 4.2 kb gene in a vector. Restriction digestion confirmed the presence of the insert. When I carried out PCR with the primers of the gene I get a band but its only 3 kb. How is it possible to get a band of smaller size using the same primer? The gene is not altered in size as I get the desired protein product. How do I optimize the PCR of the vector?
Hi Victoria,
Have you checked wether the primers encompass the full lenght of the cloned insert, just by checking the priming site on the consensus sequence? I know the question sounds silly, but I do not know whether you inherited the primers from someone else or you designed them yourself.
Hi Victoria,
Sometimes when cloning, people include some of the 5' and or 3' untranslated regions of a gene. Restriction digestion will confirm insert as your expected site but Salvador is correct in that your actual gene (from the ATG to the stop) could be much smaller. Confirm the location of your primers on your insert first. Or, another option, is to use primers (if available) on the vector that flank your insert and PCR amplify...if that product is ~ 4.2kb and your PCR product using gene specific primers is 3Kb, chances are your insert contains some additional sequence of some kind.
Is there an similar sequence (or repeat) in the insert which can be a template for one of your primers? If so, short PCR product (3 kb) will be preferentially amplified than that of long PCR product (4.2 kb). Can re-design another pair of primers for checking.
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