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HELP - Adaptor ligation to DNA not working!

Ligation

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#1 anh109

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Posted 14 January 2014 - 01:44 PM

I've been trying to do a ligation for a few months and it has not been successful. Previously, I used the NEB end-repair kit to blunt-end my sheared DNA, then T4 DNA ligase for ligation at 14 degrees for 16-24 hours with 1:10 ratio. Currently I've been using an oligo with phosphorylated 5' and 3' ends to test out the ligation, and this is also not working. I have 2 adaptors, which are both ds, and each adaptor has an overhang that is complementary to the other. Would this play a role in my problem with ligation?

 

Also, does anyone have any suggestions on how to ensure that both adaptors ligate onto the dna (ultimate goal is to have adaptor1 - sequence - adaptor 2)



#2 phage434

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Posted 14 January 2014 - 05:01 PM

I don't know what you mean by "an oligo with phosphorylated 5' and 3' ends to test out the ligation." Phosphates on the 3' end inhibit ligation, and you need a dsDNA substrate for ligation to occur. Perhaps you can provide more details about your ligation experiments so far.







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