PCR problems on high GC content gene - Trouble with Colony PCR of TOP10 transformants w/ TOPO-vector (Jan/26/2010 )

Hey guys,
This is my first post on this forum, I hope you can steer me the right way!

I have had difficulty doing a PCR on a gene from T.thermophilus both using Biomix red(Taq) and Accuzyme(Hi-fidelity). However, in the end I managed to get my band with the addition of DMSO and touchdown protocol(although I also got a non-specific band).

After gel purifying the band, I ligated it to Directional TOPO vector( pet151-DTOPO kit from Invitrogen).

I got colonies from my transformation of TOP10 cells with the insert containing-plasmid. However, when I try to check my colonies for the plasmid, the colony-PCR keeps failing. It has worked simulatenously for another gene, which is from Pseudomonas sp.101 and has lower GC content. In my colony PCR, I added the DMSO/and did not add DMSO and did the touchdown protocol, but it does not work regardless of whether or not DMSO is present.

I highly doubt my transformation has not been succesful and I think the problem is with the PCR. Regardless, can anyone tell me what are all the things I can do now to confirm the presence of insert-containing plasmid in my colonies. What things can I do to ascertain that it is the PCR and not a failed transformation? Is there a possibility that a the particular gene in the plasmid can prevent colony-PCR from being succesful, and thus require me to do a mini-prep of all the colonies to get only the plasmid for PCR detection?

Thanks a lot guys.
Please ask me for any details, i.e primers I used, more details on the pet151-DTOPO etc if required.

Sometimes colony PCR just doesn't work and you just have to go ahead and do minipreps. I'm excited when it works, but not too upset if it doesn't.

microgirl on Jan 26 2010, 10:44 AM said:

Ah I see. I figured it must be the problem. Oh well, here goes miniprep time!!!

Can you think of reasons why colony PCR would fail with a particular gene in the plasmid and not another?

Are your primers flanking the insert? If so, the control (which doesn't have the insert) should work. If the control doesn't show a band, then it will prove that colony PCR is the trouble.

predoc on Jan 26 2010, 11:08 AM said:

Ah i see, the primers I am using start from the beginning of the gene. But TOPO has sequencing primers, that flank the insert, I will try this tomorrow. Cheers.