Selection of clones after ligation? - (Oct/07/2011 )

If you are just EtOH ppting your cut oligo, there is a good chance that the ends of the ds oligo are interfering with your ligation. Each end will have a compatible overhang with your vector on one side, and a blunt end on the other. These little ds oligo "nubs" can just act as a cap to your vector, preventing proper ligation. However, since you are cutting your oligo, then all fragments (the overhangs of both ends and both sides of the insert) will have 5' Phosphates.

I do understand that you cut your vector with 2 different enyzmes, and so you think you don't need to phosphatase treat your cut vector. This is a mistake that a lot of people make. There is a difference between what theory tells you is supposed to happen versus what reality tells you actually does happen. Unless you perform a vector only ligation and transformation and get ZERO colonies on a selection plate, then there is always the risk of vector self ligation. Here is how this happens:

(1) A double cut vector, cut in the MCS, looks the same on a gel as a single cut vector. Even 10 molecules of single cut vector that you can't see will religate and transform.
(2) Single stranded overhangs are always subject to shearing or degradation, so incompatible overhangs do not guarantee zero ligation
(3) Self-ligation is always SIGNIFICANTLY more efficient than bi-molecular ligation, so Vector Only will always be favored over Vector+Insert

Therefore, if you give it the opportunity, even just a few molecules here or there, Vectors will religate on themselves and give rise to colonies on your transformation plates. This is why many people phosphatase treat their vectors, even if they should have incompatible overhangs.

If you were to start all over again, I would redesign your oligos so that you would generate the overhangs that you need by annealing, rather than digestion. I would be sure to add 5' Phosphates to the oligos, and Phosphatase treat your vector. If you don't want to re-order your oligos, you can try to gel purify your cut dsOligo on a TBE-PAGE gel or high-percentage agarose gel, as long as your extra cut ends are not similar in size. The TBE-PAGE gel will give you the best resolution and yield, but if you aren't set up to do that, then a high percentrage agarose gel (2.5%-4% NuSieve should work) is your next best bet, but just be sure you digest a lot of the oligo because your gel purification yield from QiaQuick on a 50 bp band will be fairly low.

Best of Luck.

allynspear on Thu Oct 20 12:08:15 2011 said:

Thank you so much allynspear. I appreciate your knowledge and detail answer...its really worthful for the beginers like me.

Thanks a ton
Thapa