TA cloning problem - (Jul/13/2005 )

Hi everybody,

We are trying to clone app. 500bp PCR product. We have used two different cloning kits PGEMTeasy (Promega) and InsTAclone (Fermentas). Unfortunately either we could not get colonies with insert or we get no colonies. We use Fermentas Taq polymerase to amplify our product, we gel purify it and check on the gel for quantification. We have tried different insert:vector ratios, different hosts, but still we could not optimize the technique.
Another thing is we have a sample kit from takara (Ligation kit, mighty mix), we want to try this kit as well. But, this sample does not include vector, can we use any vectors with it, like ptzr57r/t?
Since we do cloning in our lab for the first time, we do not have experience, thus your suggestions are highly appreciated.

Thanks
emel

Hey,

for TA cloning it is important to make sure you product has got A´overhangs. I you haven´t already done you should leave your PCR at 72°C for 10 minutes (after your last cycle).

Another kit that works good in our hands is "TOPO TA cloning kit" from Invitrogen. You select your colonies with blue/white selection. Just clean your PCR Product and ligate within five minutes. Good stuff.

Good luck
Cheers

Try a new aliquote or tube of ligase buffer. ATP is required for proper ligation and repeat freeze/thaw of the ligase buffer degrades the ATP.

If you don't have another aliquote, just add a bit of ATP to the ligation reation.

HTH

Jeff

Is your gel purification step necessary? If you do not have aspecific products I would just use some µl of your PCR to do the TA-cloning. If you still have primer dimers, you can remove them with any PCR-purification.

Is that possible that there is some mistake during plasmid transformation step? For example, your plate (proper antibiotics?).

Hi everybody,

We are trying to clone app. 500bp PCR product. We have used two different cloning kits PGEMTeasy (Promega) and InsTAclone (Fermentas). Unfortunately either we could not get colonies with insert or we get no colonies. We use Fermentas Taq polymerase to amplify our product, we gel purify it and check on the gel for quantification. We have tried different insert:vector ratios, different hosts, but still we could not optimize the technique.
Another thing is we have a sample kit  from takara (Ligation kit, mighty mix), we want to try this kit as well. But, this sample does not include vector, can we use any vectors with it, like ptzr57r/t?
Since we do cloning in our lab for the first time, we do not have experience, thus your suggestions are highly appreciated.

Thanks
emel