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Ligation of Blunt PCR Product


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#1 Bill_H

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Posted 30 July 2010 - 05:53 AM

Hi,
I want to join two PCR products that are blunt ended without forming concatemers. Does anybody have any suggestions?
Bill

#2 perneseblue

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Posted 31 July 2010 - 06:11 PM

Hi,
I want to join two PCR products that are blunt ended without forming concatemers. Does anybody have any suggestions?
Bill


Could you provide us with more details such as
what you want to this with ligated PCR product.
how were these PCR products generated

The immediate idea that comes to mind is to design the pairs of primers where one primer carries a unique restriction site, while the other does not.. This unique RE site does not occur in either of the two PCR products

Using BamHI and EcoRI as example

BamHI-PCR-A

PCR-B-EcoRI


Ligate both PCR products. Once ligation is complete, cut the ligated products with BamHI and EcoRI. The BamHI and EcoRI will cut any concatemers that have formed into dimers. Furthermore it will also cut any ligated product which was formed by ligation in the wrong orientation.

If you then ligate the PCR dimer fragments into a EcoRI/BamHI cut vector you can isolate your desired hetero dimer from a mixture of homodimers of PCR-A and PCR-B

If the free heterodimers are required, PCR amplify the above vector with a second set of primers that exclude the RE site that were added at the 5' and 3' end.
May your PCR products be long, your protocols short and your boss on holiday




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