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TOPO TA Cloning Troubleshooting


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#1 5280



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Posted 19 November 2012 - 05:31 PM

I'm having an issue with my TOPO TA cloning. My Top10 cells were transformed and I was able to get some good clones as confirmed by sequencing of the plasmid. However, I'm having a difficult time getting my clones from my LB plate with 100ug/ml ampicillin to grow in LB broth with 100ug/ml ampicillin. My plates have 10-15 blue colonies, many small and medium white colonies. The small colonies seem to be false positive non-transformed Top10 E. coli. Therefore, I was only picking the larger well isolated colonies. However, these colonies usually don't grow in LB broth with ampicillin. I'll pick 4 colonies from 4 plates for 16 clones but only 2-4 out of the 16 will grow in LB Amp. The package insert states to use 50ug/m ampicillin, would 100ug/ml cause the issue with the growth in LB. The plates are about 7-10 days old now. Age shouldn't be a problem, right?

#2 Trof


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Posted 20 November 2012 - 02:06 PM

100 ug/ml Amp should not be a problem, since it's commonly used, 50 may not be enough and can result in the overgrowth of wild-type clones. Also week old Amp plates properly stored should be OK.
Maybe you have a problem with the whole Ampicilin stock, or it's the insert itself that's causing it.

What vector are you using? As far as I know all TOPO TA screening vectors have also kanamycin resistence. I would switch to that. I don't recall ever having problem with untransformed clones with kan selection.

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