Struggle for constructs with pLENTI - (Oct/02/2006 )

HI
I try to ligate a 700bp insert(PCR product) into 7000bp pLENTI4 vector with a RsrII cutting site.
I have prepared well treated insert and vector, transform to Stable4 competant cell.
But the cloning is few (about 5~8).
Plasmid product from Mini prep is strange about 3000bp.

I have made sure every step (transformation,RE digestion, ) is well performance with positive control.

Would please give me some advice to work with a unstable pLENTI4 vector. Thank you!

is that plasmid miniprep DNA digested?

how exactly are you preparing your insert and vector?

I don't know what lenti plasmid you're using, but the one from Invitrogen's virapower kit is prone to recombination events between the 3' and 5' LTRs. This results in a smaller plasmid that could explain your 3000 bp product.

The plasmids with recombination grow faster since they are smaller, so select the smallest colonies for miniprep. Also, I was able to get more of the small colonies with the insert by not using blasticidin on the plates.

-ZP

I am using pLenti4 routinely and it hasnt been a problem.

My main concern would b the efficiency of the RsRII digested site.

if u use the stbl3 or DB3.1 cells, recombination shouldnt b a problem.