We designed a shRNA that in the end should have 53bp. However, when we ran on a denaturing polyacrylamide gel, the size was lower than 50. Also it shows a another band, slightly lower than the other.
We ordered a chemically synthetized shRNA, with the same sequence, and ran both on a denaturing polyacrylamide gel. Both of them show a lower size than predicted.
Does anyone have a hint of what could be the reason?
Thank you in advance!
Could you please specify how lower? Is your gel resolution sensitive enough to discriminate few PB differences? Temperature of your running buffer, the age of the gel could also induce artefacts such as gel smile etc...
Best,
Romuald
First of all, thank you for helping.
About your possible solutions, the bands are quite far from 50. The gel is a 12% denaturing polyacrylamide gel. Here I show the image of the gel where in the first lane is the one produced in vitro and in the second lane is the chemically synthsized shRNA. I wonder if it's possible that the shRNA can naturally "superfold" in itself.
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