I want to clone my gene of interest from a eukaryote.....I have a single 5' degenerate primer available to me. can I use single 5' degenerate primer from my amplification and oligo T as my reverse primer to amplify.
A single degenerated primer? A single primer cannot be a degenerated primer for generated primers are composed of a cocktail of primers.
Hello Syed...Single degerate primer means....only one ie for 5' end of the gene.....it will obviously will have cocktail of primers.
I see. Technically, the amplified gene can be cloned perhaps using T/A cloning. Blunt end ligation is also possible provided the gene is amplified using Pfu or some other high fidelity polymerase using phosphorylated degenerated and oligo (dT) primers. What I don't understand is, why these primers? If you know your gene, would it not be better to amplify it using specific forward and reverse primers?
Hi,
as Syed pointed out, if you have only one gene of interest it would probably be better to use gene specific primers. Depending on the gene they may already exist as a pre-made assay, otherwise you can somewhat easily design them yourself. Or is there a specific reason you don't want to do that?
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