Cloning problem HIV-1 Reverse Transcriptase

Sascha stumpp at rz.uni-leipzig.de
Mon Feb 16 11:38:53 EST 2004


Help !!!!
I'm trying to clone a subunit of HIV-1 Reverse Transcriptase into
pQE30 (E. coli host XL1 blue). The vector is a little bit modified so
don't wonder why I'm using Bsp1407I (Cloning strategy).
I'm trying this now for several weeks and it's still not working.
After Ligation/Transformation I always get clones but the isolated
plasmids are always too short ( no religands, no cutting with
apropriate enzymes). I don't think there's a contamination in the
water we're using.
 I know that Reverse Transkriptase is toxic for the E. coli host so
I've tried to repress expression by using 2% Glucose in the media -
nothing. Transformation using lower temperature (30 °C instead of
37°C)- nothing. I know that XL1 blue has the F' episome expressing the
lacIq that should already repress the expression.
Does anybody has experience with E. coli ABLE (Stratagene?) cells.
They should be optimized for cloning toxic proteins.

A few words about my cloning strategy:

Amplification of the insert by PCR
- Ph/CHCl3 extraction 
- EtOH precipitation
- Gel-purification using Qiagen Kit

Double Digestion of Vector(+Dephosphorylation, Alk. Phosphatase Roche)
and Insert
-Heat inactivation of both enzymes (PstI and Bsp 1407I)
-Ph/ChCl3 extraction
-EtOH precipitation
-Gel-purification using Qiagen Kit

Self-Ligation test of vector and insert performed: everything ok


Ligation 
-Vector/Insert 1:3 in a 20 uL Ligation reaction using Promega T4
Ligase and buffer

Transformation in XL1 blue (e-competent, do it yourself, efficiency
5*10_8 cfu)

Selection on LB amp (100ug/ml) Agar plates, 2% glucose.


Any suggestions ????



Best regards
Sascha


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