can colony-PCR screen for big inserts? - (Sep/04/2006 )

Hi all,

These days, I was working on cloning 3 different genes (1.5kb, 1.1kb and 1.5kb respectively) into TOPO pCR2.1 vector. And it worked fine. The next step I would do is to ligate these 3 piece together and move as a whole into my target plasmid. My question is: will colony-PCR work to screen for the big insert (in my case, 2.6kb for 2 pieces combined and 4.1kb for 3 pieces combined).

I am worring if the Taq (GOTaq, Promega) can amplify such fragment although Promega company claims it to work..

if colony-PCR is not a good way to go to, what are the possible alternatives?

thanks,,

An interesting subject to compare notes ...

I use home made taq for colony PCR and the biggest PCR product I ever made in such a screen was 2kb.

However, that screen gave we the jitters. If I recall corretly, product yields for Taq drop off a fair bit for products 2kb and up. I didn't like that feeling one bit, so I make(buy) diagnostic screening primers to keep the product size around 1kb and below.

Anyhow, my suggestion would be to do for one 4 way ligation (if your ends are incompatible with each other) into a colour testable vector, then move fused insert into target vector. It saves time and entertains your labmates. Buy specific primers for the colony PCR screen. It is relatively cheap to buy short primers.

Alternative screening methods employed in my lab are LOTS of minipreps. (48 - 72 - 144), and colony hybridisation (screens 2000 at one go - 22cmx22cm plates).

Hi,

I think it's worth a try to colony PCR your new inserts.

Or, you could just do multiple PCRs (or multi-PCR) with different combination of your readily-available primer sets, which are specific to fragment 1, 2 and 3. If orientation is not your concern (e.g. if you had used 2 REs for each ligation), that'd work fine to confirm the presence of fragment 1 and 2 (or 2 and 3; or 1,2 and 3) in the insert.

Cheers,