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Restriction digestion of direct small PCR product and cloning


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#1 ampelo

ampelo

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Posted 22 June 2011 - 05:35 PM

Hi Folks,

I am interested in modifying my vector with additional restriction sites, customized multiple cloning site,my strategy is to make two primers, the forward primers will have 5 restriction sites and the reverse primer will have 3 new restriction sites and 2 restriction sites will be complementary to the forward primer so that at the annealing step the two common restriction sites common to both forward and reverse primers will anneal to themselves and amplify each other and extend the portion which are not complementary to each other. My PCR had worked and I am getting 50 bp PCR fragment. I have a single restriction site, AVR-II on both ends of the PCR, so that I can open my vector and insert my 50 PCR fragment after digesting it AVR-II. My question is,how do I do a restriction digestion of 50 bp product? I tried to clean up with ethanol precipitation but in the process lost all of the DNA. Now I am thinking of restriction digestion directly the PCR product and ligating the digestion product directly to the linearized and dephosphorylated vector. Does anybody have a better strategy to insert the MULTIPLE CLONING SITE inside my vector? I do not want to do the cleaning up step with such a small PCR product so does anybody have any suggestions for me? Will appreciate your comments or suggestions!


Ampelo




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