Random Amplification + TA cloning = HATE - (Dec/03/2007 )
So, here's the deal. I've been randomly amplifing viral RNA, then trying to do a shotgun sequencing approach. However, I've been rather unsuccessful at getting the little bastards to clone in. I gel extract, using a Qiagen kit (to avoid alot of small clones), and I am very nice to my DNA -- I cut the gel and only stain the marker then use it to cut out my target DNA. I run a gel after eluting to make sure I have DNA still - yep, still there - then I add A's with Taq at 72C for 15 minutes. After this, I've tried both PCR purification (again Qiagen) and just proceeding to ligation without any purification -- neither of which worked. I use the Invitrogen pCR-XL TOPO kit with electrocompent cells -- and my positive control (just pUC 19 plasmid) works well so I know its not a transformation problem. Any ideas would greatly appreciated.
Are you adding dATP or dNTPs to the tailing reaction?
Hu has shown that the extended nucleotide is not always an A residue, and is dependent on the specific nucleotide present on the 5’ ends of the dsDNA.
Hu, 1993 DNA and Cell Biology 12 (8) 763-770.
Hite, J., Eckert, K. A., Cheng K. C., 1996. Factors affecting fidelity of DNA synthesis during PCR amplification of d(C-A)nd(G-T)n microsatelite repeats. Nucleic Acid Res. 24: 2429-2434.
I've done both dATPs and dNTPs, actually, niether of which worked. Thanks for the suggestion though -- I'll keep it in mind. I was doing dATPs until my boss told me to use dNTPs...