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Digestion and ligation problem! Need advice


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#1 Alkaiser

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Posted 28 December 2010 - 12:43 PM

I'm trying to ligate a 700 bp fragment into a 7 kb vector- I've PCR amplified the 700 bp fragment (which was in a 3 kb vector) with restriction enzyme sites on each end (the same sites that I made my vector have in order to ligate). When I designed my primers, I added 3 extra bases to each end after the enzyme site.

The problem is that I didn't notice that the 700 bp fragment has one of those two sites in the middle of it- about the 400 bp mark. So if I run a digestion on the PCR amplified insert, then it will cut it into a 400 and 300 bp fragment, in addition to cleaving off the very tips of the insert. What should I do? I don't really want to have to quick change the site inside of the insert, but that's a possibility- it's just time consuming. Is there a way to avoid this? If I run a digest on this insert and then ligate the 400 and 300 fragments together (95% ligation efficiency) would that be a good solution, or would the 4 or 5 bp end that was cleaved also get ligated back? (I'm hoping not!) I'm in trouble! Thanks in advance to anyone who can help!

#2 perneseblue

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Posted 28 December 2010 - 04:34 PM

Easiest and fastest solution : Buy another primer with a different restriction site that will allow you to ligate your insert into the vector and avoid cutting the insert. Primers are cheap.

The idea that your proposed would work. But you need to add your vector into the ligation mix. Attempting to religate the two 300bp and 400bp pieces together on their own will not work. All you will get are concatemers. To repeat myself, add the 300bp, 400bp and vector into a ligation in a mol ratio of 1:1:1. Transform the ligated DNA into cells. Then screen for a colony which has the two insert in the correct orientation (one fragment can now ligate in two orientation as it ends have the same restriction site). You will need to screen many colonies (~72). Colony PCR and a multichannel will help you get you your plasmid.
May your PCR products be long, your protocols short and your boss on holiday




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