4 replies to this topic

[Sharky]

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.

Edited by Sharky, 26 January 2010 - 09:17 AM.

[microgirl]

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.

[Sharky]

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

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.

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?

[predoc]

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.

[Sharky]

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

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.

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.