shRNA ligation problem - (Oct/22/2008 )

Dear all:
I have buy the BLOCK-iT™ U6 RNAi Entry Vector Kit from invitrogen company, and design shRNA seuquence about 50 bp in HPLC level.

I did the annealing, liation , and transformation following the protocol provided with the kit, but the result is: I got the bacteria clone of the positive control plasmid provided in the kits, but can not got clone of the LacZ double-stranded Control Oligo provided in the kits, and also can not get the clone of plasmid containing shRNA sequence.

For I can get the clone of the positive control plasmid, so the DH5a and transformation protocol used should be OK. And I have run the gel to check the annealing result, it also in good condition. So,
maybe the ligase and ligation buffer provided in the kits has some problem. I used ligase and buffer provide by other company, also can not get the clone.

Do anybody else experienced such things, hope your useful suggestion.

ligation of shRNA is a challenge for a normal cloner. Be prepared for some to get it work! The insert has a loop and the viral plasmid is much clever than normal dump plasmid.

It won't be easy - be sure to use high competent cells for the transformation (as efficiency is so low)- and the invitrogen cells are probably the best for it. I spent ages doing it in block-it - then ages getting my constructs through BP/LR... for no reason.. it just worked in the end.
Make sure the pLenti6 prep is endotoxin free aswell (though I think yours is the bought pre-cut version?)