Hello,

I have cloned the human Interferon gamma gene in a lentivirus plasmid. minipreps were fine. I did twice the maxi prep on it but my yiled is really poor.

I was wondering if anybody had experience with this gene and if it is toxic to the bacteria. (PS: I do a lot of maxi and used the plasmid backbone before with great results)

Thank you very much for your help

Is the backbone plasmid pLKO.1?

We do quite a lot of work with constructs from Sigma's Mission lentiviruses and find that there is often a problem growing up the maxis (although minipreps were less of a problem).

We get around it by treating the plasmid as a low copy plasmid.

I use a modified Qiagen method:

Use 20mL of P1, P2 and P3 (ie instead of 3 x 10mL)
Spin down and transfer the supernatant to 2 x 50mL Falcons
Add 0.7 volumes (fill up the tubes) Isopropanol

MIX WELL and centrifuge fast for 30-40 mins to pellet the DNA.
Remove as much liquid as possible and allow the pellet to dry a LITTLE - NOT TOO MUCH (otherwise it is difficult to resuspend)

Resuspend it in 1-2mL of water, add to this about 10mL of QBT (Column equilibration buffer) and load this on to the pre-equilibrated column (TIP 500).

Then continue as per protocol.

This just removes a lot of the protein before the DNA is added to the column and really improves the yield from almost nothing to over 500 micrograms.

Thank you so much for this tip,

I use a non commercial Lenti (with a cppt and WPRE sequence). I have used this vector before without problem for DNA and virus prep.

For endotoxin removal at what step can I add my endotoxin reagent? In the 20mlx3 mix or in the 2ml resuspended DNA?

Again, thank you very much for this helpful tip.

PS: we checked our DNA concentration by doing a miniprep on the maxiprep culture and it was good

I had a look at the endo-free maxi protocol and I think I would resuspend the pellet in 1mL water, then add about 3 mL of P1 (to get the right buffer conditions) and then add the endotoxin removal agent. After the 30 mins or so, this would be loaded on to the column and processed as stated.

I haven't done an endo-free maxi for quite a long time. Do you find that lenti titres are better when the transfected plasmid DNA is endo-free?

Thank you
Yes the titer is better; the cells look better and produce more proteins. The transfection is improved. I always do endo-free.
in labs around ours, my colleagues also do this in routine.

Thanks again for everything