Hej everyone,
I’m having trouble getting the correct sequences when trying to ligate an insert for the expression of an shRNA (that I generated by annealing two oligos) into a pLKO.1 vector.
Namely, I get point mutations, especially symmetrically flanking the variable part of the sequence.
So far I have tested around 25 Oligo-pairs which all were similar in length etc., for some everything worked fine right away and some were correct after the 2nd or 3rd try...
I have tried a slower annealing protocol (-3°C per 10 minutes) as well as repeating ligation and transformation (Stbl3).
Any ideas what could be the problem here or any suggestions on what to try?
Thanks a lot in advance!
Katharina
Did you check the original sequencing profiles before concluding that these are bona fide point mutations?
Since the mutations did not appear again in some of the constructs after repeating all steps I figured it must have to do something with the cloning itself.
Hi Katharina,
Have you checked for possible secondary structures inside individual oligos or between them? Secondary structures are very commom in sequences containing complementarity like shRNA cassettes. The mutations could be the result of the bacteria's repair systems trying to repair the mismatches in the plasmid, even for the Stbl3 that should be more flexible in this sense.
Hej Sebastián!
Thanks for this valuable hint! For now I have confined myself to sending more clones for sequencing and I got right sequences for almost all constructs! One stubborn construct is left - if the sequences of the last clones I sent in don't come back correct I will try and transform the plasmid into another sufficiently stable E. coli strain!
Thanks!
Katharina
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