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TOPO-TA Cloning PCR product


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4 replies to this topic

#1 littlecell

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Posted 06 January 2005 - 05:10 PM

B) :ph34r:
i would like to clone my PCR product into the pCR-TOPO II vector from invitrogen. using the method recommended by the manufacturer ( only AMP in the LB/agar plate), i did get a few white clones. but when i extracted the plasmid from at least 5 clones and did restriction digestion, i could not find any insert there. it's said that the effienciency of TOPO CLONING is very high. the pCR-TOPO II vector is AMP and KANA double resistant. should i add KANA into the LB/agar plate when i steak the transformant? could anyone give me some advice on TOPO-cloning? appreciate your suggestion!

Edited by bioforum, 06 January 2005 - 05:37 PM.


#2 paulina

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Posted 06 January 2005 - 05:46 PM

One antibotics is enough for selection.

Did you also see blue colonies? Is your AMP freshly prepared? The kit can guarantee 100% success. Most problems arise from plates, antibotics, beta-gal, IPTG, and less likely ligation reaction.

#3 littlecell

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Posted 07 January 2005 - 09:59 AM

thanks your reply very much! yes,i did got 2 blue clones with about 50 white clones on one plate. i picked 4 white clones to culture ( only AMP select) and extracted the plasmid. to be honest, i used the plasmid kit from sigma and got a lot of genomic DNA contamination and almost no plasmid! it's very weird. did the sigma plasmid kit not work? by the way, someone else told me that ONLY AMP is not enough. so i cultured the previouly storaged white clones in LB medium containing both AMP and KANA. they growed very well.

#4 Microman

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Posted 07 January 2005 - 11:52 AM

This may help, i have used the kit in the past.

I used only AMP selection.

The fact that you are seeing colonies suggest one of two things, that the transformation was successful as it is the plasmid that confers antibiotic resistance. At one stage i did have a problem were i was getting colonies but wasnt finding plasmid in the TOP10 it turned out that there was a problem with the antibiotic. But when i made fresh antibiotic it fixed the problem.

In terms of efficency i have found between 3&50 colonies on plates LB AMP plates and in some instances i have found a lot more.

#5 mikew

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Posted 07 January 2005 - 02:34 PM

In theory, AMP is definitely enough. In practice however, I found that if I didn't add KANA I got alot of false positives like you did. I don't know why. AMP alone should be sufficient. I suspect an inherent resistance in these bacteria but who knows?




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