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1-2kb PCR products TOPO cloning troubles - help (Oct/01/2004 )

Well here's my problem:

I'm trying to clone 3 differents PCR products into PCR4-TOPO. Their length vary between 1.3 kb and 2.5 kb, wich is not that huge I think.
The thing is I used Pfx (proofread) to amplify the fragments, then added the A overhangs by adding dNTP and Taq + 10 min 72C. I then used the max amount of PCR products to perform the ligation reaction, and so forth : I made everything advised to get results, but it doesn't work!!!

Should I purify the PCR products after adding the As? Should I use more vector in the ligation reaction?
Should I...? I don't know what to do!!!

If you know how to solve my problem please tell me...
Thank you very much

-julie-

Hi Julie,

Have you included any controls in your experiment? Controls are essential for ligation and transformation troubleshooting as has discussed here many times. You can use intact plasmid such as pUC which is usually included in cloning kits as positive control. If you could not see colonies in control transformation, the problem may then be related to your competent cells.

I think you should purify your PCR reaction before ligation.

How about including a Taq amplified PCR product as a control for the effciency of adding a "A" by the Taq following Pfx amplfication?

-pcrman-

Hello and thank you fo your help!

As a matter of fact I made some control reaction, ie I cloned another plasmid (=> my competent cells are ok); and I couldn't clone the fragment after Taq amplification either (ok this I should have told sooner... rolleyes.gif ).
So I'm a little desperate! But I'm gonna try purifying the fragment before ligation (and maybe saying a little prayer or something!?!)

Thanx again!

-julie-

laugh.gif It works!! Petri dishes full of bacteria!!
Thanx again and again!!

-julie-

congratulations!

Actually I often spend a sleepless night after doing a transformation and fear I may not see those lovely and shiny colonies the next morning.

-pcrman-