cloning problems - (Mar/18/2005 )

Hi everyone,


I'm getting crazy trying to insert my cDNA (5kb) into a mammalian expression vector (a pBABE retroviral vector), which size is approx 5.5kb.

The situation is the following:

destination vector cutted with two different REs (1 sticky and 1 blunt end, the choice is a forced one because several of the restriction sites in the MCS are also present in my cDNA)
insert cutted with the same REs as the destination vector.

The insert and the linearised vector have been purified on gel following electrophoresis and ligation performed 4h at RT (but also ON at 16°C).
Ligation products transformed using either XL1 blue and DH5a. V:i ratio was 1:3

The outcome is non colonies in the v+i + ligase plates, no colonies in the v + ligase plates, colonies in the single cutted + ligase vector (both the blunt-ended and the sticky-ended)

The same problem occurs when cloning the cDNA into a different mammalian expression vector, pCMVTAG2B from stratagene (4.6kb), with the same directional-cloning strategy.

Let me say that others have cloned that cDNA so that I think there would be no problems of toxicity in the bacteria host and I have been able to clone the same cDNA into pBS either under the T3 or T7 promoter.

There is anyone that could give me clues...I really need to clone my cDNA into those vectors...aarghh!

Thanks in advance,

Pietro

hi
my colleague did have the same problem as you insert in pBABE with a insert size equal of pbabe.
Finally she gets her clone with many experiments and try different conditions (i mean ratio v:i)
there were : v:i 1:3, 1:5: 1:1, 3:1 and 5:1.
i on't remember which one was the "succeding" condition.
fred