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I'm just about to start working on a TOPO cloning after I couldn't get it to work with standard restriction/ligation. Does anyone have any tips for TOPO cloning?

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closed as not constructive by Mad Scientist May 1 '12 at 8:21

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Can you provide the protocol you actually used so that we can better identify what could have gone wrong? How old is the kit? Was it stored correctly? Which cells are you using for transformation? –  leonardo Apr 30 '12 at 12:08
    
Ah sorry I should clarify, I first tried the traditional approach to cloning by inserting restriction sites either side of my gene via PCR then double digesting the PCR product and target vector. Following double digestion I then attempted a ligation but I nor my colleagues could get any positive transformants. So I am moving to a different method of cloning using the TOPO kit and I am wondering if anyone has any pointers before I begin using it. It is the pET101/D-TOPO kit. –  Ben Apr 30 '12 at 15:47
    
This may fall under the category of a discussion. I will say that you should make sure that you have the right polymerase if you're doing TA TOPO cloning. –  bobthejoe Apr 30 '12 at 18:09
    
And if you're troubleshooting, I would check the activity of your ligase. If your digestions are working then your doubly-digested PCR insert will ligate with each other. If not, you may have some issues with your restriction enzymes. –  bobthejoe Apr 30 '12 at 18:11
    
thanks @bobthejoe. I suppose you are correct it is more of a point of discussion, apologies for that. The original cloning underwent trouble shooting and the problem could could still not be solved. This question can be closed. –  Ben Apr 30 '12 at 18:25
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up vote 5 down vote accepted

I have lots of experience with the TOPO TA kit which is very similar to the kit your using. Here's a link to the TOPO Directional cloning product page which shows a schematic of the enzymatic ligation.

From your comments, you said you have doubly-digested the PCR product. This is likely your point of failure unless you are absolutely confident that your enzymes work at blunt ended DNA (which I can almost guarantee does not work). Restriction digestion of PCR products will only work if there are at least a few bases on either side of the recognition site so that the restriction enzyme is able to "grab on" to the DNA. What you are mixing instead, is blunt-ended PCR product DNA + cohesive-ended vector. This will not work under any circumstances as is.

There is a simple solution.

Add 3'-A overhangs to your PCR product directly. The back of the TOPO manual tells you how to do this as well as many other sources. Ligate this using your TOPO kit to produce a shuttle vector that you can then doubly digest your insert out and gel purify it. Then you may ligate this purified, digested DNA with your previously digested destination vector.

Note that in my hands and in others in our lab, topoisomerase is unstable and has a short half-life. It is especially sensitive to temperature changes (warming from -20°C to 4°C) which you wouldn't expect since it is designed to work at room temperature. The vector is not affected, but without a working topoisomerase, the ligation step does not occur and the DNA does not transform.

As an aside: If you plan on doing more cloning in the future and need to replace the kit, I would recommend getting the basic TOPO TA cloning kit, clone your PCR product (with 3'-A overhangs) into the vector, transform, pick white/light blue colonies for screening then cut out from the TOPO vector (the shuttle vector) for downstream applications. The fact that it is directionally cloned or not into the TOPO vector is of no consequence unless it also happens to be your destination vector.

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great advice thank you. –  Ben May 1 '12 at 11:34
    
You're welcome. :) I should mention that since you have the directional TOPO kit, you'll need a different ligation method that does work with either blunt DNA (eg, a blunt cut plasmid) or 3'-A overhangs (the original TA kit, pGEM-T, home made T-vector, etc). The addition of those extra bases in the directional kit will also hinder direct ligation. –  leonardo May 1 '12 at 14:55
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