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?
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