gRNA CRISPR cloning vector help.

[Regina Husman]

Hello everyone, I am desigining the overhangs for the plasmid linked below.

http://www.addgene.org/static/data/plasmids/52/52963/52963-attachment_mRK7DHmyjqVo.pdf


The plasmid indicate that the restriction cloning site is : "bsmbI", however in their cloning protocol, their designed overhangs does not match the bsmbI cut site.  Anyone have ever designed oligos for this plasmid ?

Thanks!

[Adam Freund]

Hi Regina,

Both BsmBI and BbsI (used in other backbones from the Zhang lab) are types of restriction enzymes that cut several nucleotides away from their recognition site.  This is actually a great feature - it allows directional cloning with a single restriction enzyme.  However, it means that the overhangs you need for oligo annealing don't match the BsmBI recognition site; instead, they match the overhang that is generated after BsmBI digestion.  As a side note, this leads to the BsmBI sites being destroyed once you ligate your oligos.

Hope that helps!

[Agnieszka Chronowska]

Hi Adam,

I was just trying to find out whether BsmBI site is destroyed after cloning an oligo. Your post answered my question. This would mean that I cannot take a GeCKO pool library to remove oligos that are there to recover the vector to clone another oligo that I need.

Thank you

Aga