I’m looking for some help troubleshooting my cloning process. I’m trying to ligate a shRNA into a plasmid (pWALIUM20, ~10Kbp) but have been unsuccessful many times. I’m using a double digest (EcoRI-HF and NheI) to create sticky ends and use CIP to dephosphorylate those ends when digesting. I’ve mainly used the T4 Ligase ligation protocol from NEB, but have also tried alternatives. I’m using 0.02 pmols of plasmid and 0.06 pmols of insert in my ligation reaction (Insert:Plasmid). I’ve double checked that my shRNA strands are annealing together and my transformation positive control looks good. Once I transform, pick colonies, miniprep, and sequence the insertion site of my plasmid, it looks like some of the plasmid is degraded and is reannealing to itself. Would anyone have any further troubleshooting suggestions? Or tips on running a successful ligation?
1: you only use the cip for the plasmid, right?
2: which E.coli do you use, if I recall correctly, plasmid with shRNA need a special E.coli, not dh5 or top10.
I have done shRNA cloning in pLKO.1 vector. I hope that the expected size of your oligo strand is 60 - 70bp. Use HPLC purified oligos instead of desalting ones. Because in the first case, you could get full-size oligos, not contaminated ones. Follow Takara shRNA cloning procedure. It works very well. People often think that any special type of ligation of transformation is required for this type of cloning. But that is completely wrong. Your oligo quality should be good. Use Stbl3 comp. cells for transformation.
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