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problems with topo ta cloning

problems with topo ta cloning - Molecular Cloning Forum

problems with topo ta cloning - 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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Old 10-17-2010, 11:36 PM
Pipette Filler
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Default problems with topo ta cloning



Hi guys,

First post here and in a bit of a bind. I have been trying to clone a 3kb gene into the topo vector. I did the pcr, added 0.2ul for taq to my 50ul reaction to add the adenine overhangs (i had used a high fidelity enzyme for my pcr), purified it, set up a topo cloning reaction using 4ul of pcr product and 1ul of vector. I got good number of colonies on carb plates but when I miniprepped and ran on a gel I only saw bands corresponding to the topo vector. Any idea what's going on? The concentration of my pcr product was 38.6 ng/ul.

Does the ratio of insert to vector matter a great deal? That's the only thing I can really think of. My mentor didn't specify the need to calculate, he just told me to use 4ul. Do you guys have any other ideas as to what's going on?
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Old 10-19-2010, 09:25 PM
TGS TGS is offline
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Default Re: problems with topo ta cloning

In experience the efficiency of Topo (at least for TopoXL, didn't tried another Topo-System yet) drops with increasingly length of the insert.

In our lab we normally prepare our PCR products for Topo as follows:
8 uL purified PCR-Product
1 uL Buffer
0.5 uL dATP
0.5 uL Taq-Polymerase
20 min at 72C.

Afterwards we use 4 ul from this solution for the cloning itself (or 2 ul + 0.5 ul Topo, since 1 uL is sufficient for Transformation via electroporation without prior dialysis).

Another point is that you should definately purify your PCR via gel electrophoresis. You'll always have some small fragments as unspecific side products, even if not visible on the gel. These will have a much easier time getting in the TOPO vector than your 3 kb Insert.

Also check the concentration of your DNA after purification. In my experience with TOPO XL it should be at least 15-20 ng/uL (more could probably be even better) for a successful TOPO cloning with such a big insert.
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Old 10-19-2010, 11:40 PM
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Default Re: problems with topo ta cloning

Will definitely try to implement some of your suggestions today. Thanks a lot!!!!
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