We are trying to clone the 60bp shRNA related to a gene in the PCDH vector, but unfortunately we do not see the desired band in PCR. The annealing step was performed with the usual protocols. However, after the annealing step, the annealing product showed a 30bp band on the 4% agarose gel. Considering that almost the entire sequence of oligos is hairpin, is it possible that the annealing step was not done correctly? Or is the shRNA cloned but not visible in PCR because of the hairpin structure?
I wouldn't worry too much about the apparent size of such a small fragment on an agarose gel.
When annealing, at the end, make sure you let the sample cool down slowly at room temperature. Also, don't forget to phosphorylate the resulting product (I use T4 PNK) and de-phosphorylate the vector (with alkaline phosphatase; reduces background of negative colonies). For the ligation, use a very high insert-to-vector molar ratio (I typically use 20:1, rather than the usual 3:1), as it helps when the insert is so small.
I hope this helps!
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