Dear all
I cloned an sgRNA library into a pLenti plasmid backbone (puromycin selection marker) by golden gate ligation and transformation. However I cannot produce any virus from the library (repeated it multiple times). Surprisingly from the plasmid backbone without the sgRNAs I could produce virus. I sequenced the extracted plasmid after golden gate ligation and transformation to see if there are any errors in sequences but found none. Anyone has encountered a similar problem and know why this is happening?
Hi Dharanija
Have you checked your library by restriction digestion? Most likely the lentiviral backbone has collapsed and it won't look anymore as the empty vector control. I had this happening in shRNA cloning in pLKO occasionally, Sanger sequencing working but no virus production and a significant reduction in the whole plasmid size by restriction digestion. Lentiviral backbones tend to recombine in bacteria, resulting in non-functional byproducts. You should use Stbl2/Stbl3 cells and/or grow the library at 30 C to avoid recombination.
Hope it helps
Francesco
Hi:
Just wondering how do you know the virus produced or not? PCR? Western blot?
Is it possible the gRNA is toxic and kills the cells?
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