inserting shRNA vs full gene into vector ? - (Sep/06/2006 )
Help ! Calling all experts !
I've had a lot of success inserting 60bp shRNA sequences into a vector using enzyme digestion of vector & corresponding overhangs on shRNA oligos. Using 1:10 ratio with T4 ligase I get reliable inserts.
However, when I use a similar tactic with full open reading frame sequences into the same vector (600-900bp)...amplified with primers to give overhangs, I don't get any colonies. The negative control ligation is blank so no evidence of recircularisation (I CIAP treat the vector after cutting).
I'm not so experienced in this & figured that I could just run the same protocol but guess not now.
any help would be great.
some more info...
BamH1 & EcoR1 cut
plasmid 6000bp
inserts 600-900 bp
currently done 1 hour at rt ligation 1:10 ratio (vector:insert)
did u have a few extra bases on each end of primer for proper digestion of PCR product ?
thanks, I didn't digest, I amplified from an existing vector using two primers that would give overhangs complimentary to the cut plasmid ends.
I would suggest digesting it, as u can not b sure of the ends, if they will b sticky.
ok, will do, thanks a lot !
...but how would that work ?
I thought a restriction enzyme needed it's site plus a few bases either side of it to cut. My product only has the site, and hopefully just a single-stranded overhang as well...
Some REs can cut at DNA ends. I'd recommend looking at the NEB chart corresponding to your enzyme to see if this is possible.
Enzyme digestion of your PCR product will likely be necessary in order to clone it into the digested vector. I believe your product doesn't have sticky ends, but instead is double stranded throughout, with an added 3' A overhang if you used Taq polymerase. While your primer design would've given sticky ends for the first couple of amplified strands the exponential amplification should create double stranded product at the ends.
I think your vector is not a vector for TA cloning. Your primers won't make PCR product with sticky ends compatible with the vector's Bam H1 & EcoR1 site. You have to digest it if those restriction sites exist in your primers.
Quite true, U have to digest it else u will have blunt ends or uneven ends in ur product so unless u have a TA vector, u should digest to insert ur product.