TOPO TA Cloning Reaction Problem

[skyler00]

I'm having a problem with cloning reaction.
I've tried 5 times using the Invitrogen kit but still I don't get any colonies on the amp plates. I got strong and clear band when I ran a gel so I don't think PCR product is fine. But even though I followed Invitrogen instruction, I don't see any colonies. I've tried to optimize the cloning reaction like extending final extension minutes, incubate the tube longer, etc...
Can anyone help me with this issue?
Thank you!

[Aurora Borealis]

Do you have fresh agarplates? We get problems with our colonies as soon as the plates are more than 1-2 weeks old.

[wolfman]

are you sure you used a polymerase which will add the extra AAAs onto your product so you can TA clone this into the vector.

[Aurora Borealis]

Originally Posted by wolfman

are you sure you used a polymerase which will add the extra AAAs onto your product so you can TA clone this into the vector.

Good point, Wolfman!

[clevermizo]

There are two basic reasons why this fails:

1) You have used a proof-reading polymerase. (You must either use Taq to perform PCR, or treat your PCR product with Taq in order to get 3'-A overhangs).

2) You have too much PCR product in your reaction (Optimal molar ratio for TOPO reaction is 1:1 TOPO vector:PCR insert. If you exceed this ratio by a large amount, you will end up with linearized strands which cannot be amplified in bacteria. Although Invitrogen does not tell you this unless you ask, the 3.9 kB pcr2.1-TOPO TA comes as a standard 10 ng/uL concentration.)

That said, when done right, TOPO-TA works like a charm. We use it in our lab for all of our cloning applications as a shuttle vector.

[hcarre]

I am having the same problem as Skyler00, although i am getting colonies. However, the sequencing data shoes nothing. I think the AT overhang may be the problem. How do i treat the PCR product to achieve the correct overhang?

[bentelizabeth]

The TOPO TA kit product insert has a protocol in it for adding A overhangs, as does the product insert for the pGEM-T kit from Promega. Of the two kits, pGEM-T has never given me any problems and I'm having all sorts of trouble with TOPO-TA. I'm using a DNA polymerase that should put A overhangs on my products, but I haven't been checking the molar ratio of insert to vector since with pGEM-T this was never a big problem.