Hi
We have cloned our sgRNA into BbsI site of CRISPR vector pSpCas9(BB)-2A-Puro (PX459) V2.0. Digestion with Age I and BbsI showed most of clones have insert (no 1 kb band compared with empty vector); however, when we wanted to verify these clones by sequencing using Hu6 promoter, none of them was working, either gave unrelated sequence or totally could not read.
Anyone faced the same problem?
Thank you.
I'm just about to sequence mine using the LK01_5 primer within the H6 promoter and will let you know how it works. I have a related question however. A previous colleague designed the target sequence inserts for px459 using overhangs that don't match the overhangs suggested in Fig. 4 of the Ran FA et al. 2013 paper. These overhangs are supposed to be complementary to the overhangs at the ends of the px459 plasmid after restriction with BbsI. The Ran et al. paper was using version 1 of px459. Does anybody know if this part of the px459 sequence changed between version 1 and version 2?
Sequencing over the insert region worked fine with the LK0.1_5 primer (sequence on the Addgene website).
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