Topo-Cloning over 3 kb - (Nov/22/2005 )

Hi there!
Has anyone ever Topocloned a fragment of 3 kB or bigger into an Topo-Cloning vector? I tried now for three times to clone in a 3 kb pcr fragment into PCDNA5FRT-To_Topo but it didnt work. I tried both Taq and Phusion-amplified pcr Products. An disadvantage with phusion is that you have to add taq and dATP afterwards and extend for further 15 min at 72 degree to get suitable ends.
But with taq I fear to get unwanted errors due to its high error rate (2.2x10-5). Has anyone ever made a big pcr fragment (3kb or bigger) with taq ? Did you have any errors in your pcr product then?
Thank you for any answers!
Katharina

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Someone in this forum once gave me a paper that described the importance of primer ends for the addition of A-tails to your PCR product. I wonder if it is possible that your PCR product is not sufficiently A-tailed to allow efficient ligation into the TOPO vector. Have a read of this paper (Brownstein, 1996 - BioTechniques 20: 1004). You can add a 5-6bp sequence to the end of your primer to increase the level of A-tailing, which may improve your ligation.

Also, what I don't understand is your reluctance to use Taq after ampilfying the product with Phusion. If the product is already amplified with a proof reading polymerase (which doesn't add an A)....adding taq afterwards shouldn't add error to your product. The way I understand it is that it will add the A to the end of the PCR fragment. Hope this helps.

M.

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You can easily do topo-ta cloning with enzyme mixes, that contain taq and a proofreading enzyme (most suppliers have at least of these 'high fidelity enzyme mixes'). I've done it with PCR-products of more than 3 kb. One thing you better do is make sure your PCR product is nicely purified (i.e. no primer dimers in you topo-reaction).

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hi
i amplified with a proof redaing enzyme a 7kpb fragment.
I check on gel efficientamplification and specificity.
Then add 1µl of Tas Polymerase and incubate at 72° for 10'
IMMEDIATELY after start tht topo reaction
That worked for and i have roughly a 11kbp plasmid showing efficient insertion of my 7kbinsert
fred

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Hi, Fred

I just wonder why you point out that the topo reaction should be started immediately after adding the 3'A ends... (I'm worried since I incubated my PCR product with Taq polymerase today at 72° for 10' but then placed the sample at 4° since I didn't have time to finish the process... What could happen to my sample...? By the way, my PCR product was made with Pfu, then purified before the addition of Taq polymerase and dATP).
Sonja

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there is a possibility that the 3'A ends start to be degraded at 4°C. And as 7kb is quite tough for a topo Ta cloning it's for enhancing chances to obtain good Topo reaction. General guideline is rather leave the pcr product without 3'A ends and do the Taq-assisted-3' A elongation than do the addition of A and leave the product overnight for ex...


i do not purify my pcr product. I just check on agarose i have the specific product and add directly 1µl of taq.


One more thing : i have better results on electroporated bacterias than with the Top10 provided.

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