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TOPO TA Cloning Reaction Problem

TOPO TA Cloning Reaction Problem - Molecular Cloning Forum

TOPO TA Cloning Reaction Problem - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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  #1  
Old 06-30-2010, 05:14 PM
Pipette Filler
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Default TOPO TA Cloning Reaction Problem



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!
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Old 07-05-2010, 12:55 PM
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Default Re: TOPO TA Cloning Reaction Problem

Do you have fresh agarplates? We get problems with our colonies as soon as the plates are more than 1-2 weeks old.
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Old 07-06-2010, 03:54 AM
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Default Re: TOPO TA Cloning Reaction Problem

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.
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Old 07-06-2010, 08:57 AM
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Default Re: TOPO TA Cloning Reaction Problem

Quote:
Originally Posted by wolfman View Post
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!
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Old 07-09-2010, 09:53 PM
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Default Re: TOPO TA Cloning Reaction Problem

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.
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Old 08-12-2010, 07:41 AM
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Default Re: TOPO TA Cloning Reaction Problem

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?
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Old 10-01-2010, 07:16 AM
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Default Re: TOPO TA Cloning Reaction Problem

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.

Last edited by bentelizabeth; 10-01-2010 at 07:24 AM.
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