I have troubles lately with subcloning using ds-oligos. I want to insert a targeting sequence (68 pb) in a vector containing EYFP. So I ordered oligos with the sequence and cohesive ends for BamH1-Not1. I did the annealing of the 2 oligos, opened my vector with BamH1 Not1, did the ligation, got colonies and by restriction, I could check that the sequence was inserted. So far so good. Then, I sent 3 colonies to sequence, and all have mutations in the targeting sequence (t becoming c), at different places for the different colonies. The EYFP is perfect. What can happen?
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1 reply to this topic
Posted 24 June 2011 - 06:28 AM
Oligos are highly impure. Usually this matters little, but when you are trying to build or mutate a construct, it can cause a lot of problems. You can pick more clones, or purify the oligos prior to using them. It is also possible that you are selecting against the correct sequence for some reason.