Primer concentration for cDNA synthesis using GSP - (Sep/26/2012 )
I am trying to clone a 3.6 KB gene from the cDNA using a GSP. I am using the Superscript III enzyme from invitrogen and they specify using 2 pmol of GSP. So I made a 0.1 micromolar stock and used 1 microlitre for priming the cdna synthesis rxn. However the gene did not amplify. (I am going to use internal primers to check if atleast 1-2 kb of the gene amplified!) Does anyone have any experience or recommendations for using a particular primer concentration which works?
Thanks in Advance,
0.1 uM/l = 0.1 pM/ul...you didn't use enough primer.