problems cloning into plenti6/V5 lentiviral vector - (Aug/12/2005 )
Does anyone have any advice on cloning into lentiviral vectors. I know its unstable due to repeat sequences and although I have used invitrogen's specific Ecoli strain (stbl 4) I keep getting back only vector or rarely a potive for my insert but into a noticeably smaller vector. Its not a huge insert, ~900bp, or is it? Double sticky ends, nothing fancy. Control ligation colonies are negligable and lots of colonies on my ligations, but all backbone vector or weird stuff of lots of different sizes.
Have tried using different sites and even blunt-end cloning but that didn't work........ha ha.
any offers?
Have tried using different sites and even blunt-end cloning but that didn't work........ha ha.
any offers?
Hi Dr. JJ,
a little bit late, but I hope it still can help you.
do you have pLenti6/V5-DEST or TOPO vector?
I worked with pLenti6/V5 TOPO and I had no problem. But this vector use TOPO cloning and not restriction cutting. According to manual, DEST should be used with Entry vector (to get insert into Entry vector you sould use also TOPOcloning and and you should perform LR recombination o get insert to pLenti). So actually no system with pLenti6/V5 use restriction cutting but both use TOPO cloning. 900bp fragment should be no problem. I cloned 3kb without any difficulties.
Good luck.
mabacil
I had some difficulty with the directional topo cloning into this vector. You need a good proof-reading enzyme for the blunt-topo cloning. If recombination was an issue you would have a smalled plasmid size due to LTR-LTR homology in the vector. Since you are recovering a vector the same size, it sounds like cloning efficiency or methodology is your issue.