Success with Invitrogen's pLenti6/v5-DEST? - (Jul/15/2008 )

I have been working with Invitrogen's BLOCK-iT Lentiviral RNAi expression system (Gateway) and failed to get reasonable high titer of lentiviruses. I am losing confidence in this vector. Just want to know if this system works.

Thank you very much in advance.

Jia

-wangj-

QUOTE (wangj @ Jul 15 2008, 05:23 AM)

It works for me and my colleagues.

There are several things that may be going wrong.

I suggest a troubleshooting of your technique to make sure you don't throw away pellet rather than the supernatant!

-cellcounter-

Thanks for giving me the big relief.

I just collect supernatant and use it directly without concentration.

Should I collect cells and do freeze and thaw cycles like preparing adenoviruses?

I didn't sequence the vector, however, the vector has right abx resistance and gives bright GFP expression in 293FT cells. So I assume the vector is correct.

I tried Invitrogen's ViralPower and Clontech's High titer packaging systems following their instruction, which use supernatant, without success.

I would very much appreciate your further help.

Best regards,

Jia

-wangj-

QUOTE (wangj @ Jul 15 2008, 09:15 PM)

We typically concentrate the lentivius sup 10X irrespective of what titer the sup has and what lentiviral system we used. May not be necessary in all situations, but well, we just do it as a matter of course.

-cellcounter-

Thank you, Cellcounter. ,

Concentration may be my solution to this problem.

By the way, do you sequence or enzyme cut the vector after the Gateway recombination to confirm you have the correct vector?

Best wishes,

Jia

-wangj-

QUOTE (wangj @ Jul 16 2008, 07:19 AM)

Thank you, Cellcounter.

,

Concentration may be my solution to this problem.

By the way, do you sequence or enzyme cut the vector after the Gateway recombination to confirm you have the correct vector?

Best wishes,

Jia

Both. We sequence only the region surrounding (and including) recombination, but do an extensive RE panel.

-cellcounter-

Thanks for the information, Cellcounter. :rolleyes

I may need to go back to check the vector.

Jia

-wangj-