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Dimerization of PCR product

cloning genetics molecular biology

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4 replies to this topic

#1 awan

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Posted 04 October 2013 - 06:44 AM

Hi Experts

 

I have a very strange problem. I did colony PCR with KAPA mastermix (annealing temp. 68C) to confirm my transformation recation. I was expecting a fragment of 1.5 kb or nothing on the 1% agarose gel. But I get a band of about 3 kb. it seems like a dimer of PCR product. is it possible?

Kindly need your help.



#2 jerryshelly1

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Posted 04 October 2013 - 09:04 AM

Is this a transformation reaction from a previous ligation? Did you use two independent RE sites when you ligated your insert into your vector?

 

Colony PCR is notoriously bad because of its high generation of false positives.

 

Where are your primers annealing for your PCR reaction, i.e. both on plasmid, one plasmid + one insert? 



#3 thefallengrace

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Posted 04 October 2013 - 10:01 AM

try double digestion, to excise out the insert. It is much more definitive than colony pcr



#4 phage434

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Posted 04 October 2013 - 11:15 AM

Most likely you have a repeated insert. Did the insert have identical restriction sites at each end? A dimer could easily form. Even if not, a dimer could form with A-B to B-A ligation of the insert, followed by insertion into a single cut vector cut at A. This does not normally happen unless the ligation happened with very high insert concentrations.



#5 awan

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Posted 04 October 2013 - 07:32 PM

I transformed pDONR221+insert to top10 competent cells. PCR was done with gene specific primers containing NCO1 and BspT1 overhangs.







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