Cloning of long gene - (May/02/2006 )
I am in process of cloning about a 4kb gene from cell lines. Please help with recommendations and dos and donts. I am using Superscript III for cDNA and Thermoscript for PCR but so far I am not yielding good results. I have made primers flanking the CDS, I hope that should not be a problem after cDNA synthesis.
"flanking the cds"?? you don't mean outside, do you? if your primers don't correspond to the sequence in the final cDNA, then you won't get a product....
have you tried internal primers to be quite certain you're getting the gene you want when you make your cDNA?