I am trying to insert a 60 bp shRNA into the pLKO.3G plasmid (variant of pLKO.1), yet nothing is working.
I digest with EcoRI and PacI, anneal the oligos at 95 for 4 mins then 70 for 10 mins, then cool down to room temperature gradually. And I do quick ligation for 10 mins at room temperature, and transform into HB101 competent cells. However, nothing is growing on the ampicillin plates. Any suggestions?
I followed the protocol they have on Addgene, but it did not work.
https://www.addgene.org/14748/
I suspect one of the active components in the process is defective. Either the digesting stage or one next to that. Contact the kit manufacturer for assistance and perhaps a replacement kit.
Well I have tested both REs I am using separately, and they are linearizing the vector.
As for the annealing buffer, I use the 10X NEB Buffer 2.1. This is what they recommended on Addgene.
As for the transformation, I am doing a 10 ul ligation reaction, and using around 5 ul for transformation, and leaving the rest at 4 degrees overnight.
That's way too much DNA for the transformation. Dilute your ligation 1:5 in water. Use 2 uL of that dilution.
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