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How to prevent concatemerization during ligation reaction?


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#1 UCI_StemCell

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Posted 28 November 2010 - 06:27 PM

I recently realized that the gene I am trying to clone has a restriction site for one of the two restriction enzymes I am using to clone right in the middle of it! If I go ahead and digest anyway, and separate the fragments on a gel, can I gel extract the two segments of the gene and ligate them together without forming a long chain of multiple fragments? I am using SacI & XbaI to clone, however there is a SacI site ~600 bp into the gene. After running on a gel, I would have a 600 bp fragment with a SacI sticky end on either side and a 1.1 kb fragment with a SacI sticky end and an XbaI sticky end. My idea was to use the Quick Ligation Kit from NEB to ligate these two fragments together to form the 1.7kb fragment of interest, run on a gel, extract and ligate into the vector backbone I wanted to originally clone into. However I am concerned that I will have multiple 600 bp fragments ligating together because they would be compatible at the SacI site on one side...can I vary the ligation time to prevent this from happening? Or is there another strategy that I should use?

I was also thinking of using phosphatase to remove the phosphates on the 600 bp fragment to prevent it from self-ligating, but after forming the 1.7 kb fragment with the 1.1 kb piece, it would be missing a phosphate on the 5' end for ligation into the vector...

#2 UCI_StemCell

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Posted 30 November 2010 - 05:40 PM

No suggestions on this one?

#3 HomeBrew

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Posted 30 November 2010 - 08:05 PM

Just recover the fragments after digestion and clone into your vector in a three-way ligation. You can check for an insert in your clones by PCR using a primer that anneals to the vector on the XbaI side and one that anneals to the insert within the SacI fragment. That way you can confirm the insert's presence and the orientation of the SacI fragment simultaneously.




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