pSUPERIOR cloning - please help! - (Jun/23/2008 )

Hi everyone,

So I am trying to create an siRNA system using the pSUPERIOR vector. I have my oligo inserts which have BglII and HindIII restriction sites (upon successful cloning, the BglII site is destroyed) and I anneal them step wise at 94oC for 4 mins, 80oC for 4 mins etc etc all the way down to 4oC. I then phosphorylate the annealed oligos and use a nucleotide removal kit to produce the oligos I use in my ligation. With the pSUPERIOR vector, I linearise using BglII and HindIII before CIP treatment and then gel purify. I then perform a ligation O/N at RT using 5ul of annealed oligos, 1ul of pSUPERIOR, 2ul of ligase, 2ul 10x ligase buffer and 6ul water. I then digest with BglII to reduce background and transform. I get colonies (about 4 or 5) which before CIP treatment and oligo phosphorylation used to be 100s of colonies. However, when I prep the colonies and digets using HindIII and EcoRI and run on an agarose gel I see the 5kb original vector and no insert. According to the manual, a successful insert should be 60bp bigger than a negative clone (around 200bp) but I cannot see a 200bp fragment even as a negative. DO you think it is my ligation or my digest with the enzymes before I run my purified DNA on a gel that is the problem?

Any help would really be beneficial!

Thanks

Hayley

Try a different set of screening enzymes. That should confirm whether it is the digest or the ligation that is not working.