We have repeatedly tried to klone two fragments of Strep.sobrinus
OMZ176 DNA into the Promega lambda-gt11 and the lambda-GEM12
vectors. The fragments are ca.4kb EcoRI digested and ca.12kb BclI
digested respectively. This is a brief description of our
protocol:
1) The fragments are separated by agarose gel electroforesis
(Bio-RAD agarose, Ultra Pure DNA grade). Fragments of the
desired size are electroeluated (ISCO Little Blue Tank with
Nanotraps). The eluate is cleaned (twice!) and concentrated
with GeneClean. The concentration of the samples is
controlled after each GeneCleaning by dropping 1fl onto
nylon membranes, staining with ethidium bromide and
comparing with known standards.
2) Ligation (both vector systems) and partial fill in reactions
(lambdaGEM12) are performed as described on the Promega
manual, except that we substituted phenol extractions with
Gene Clean.
3) We have packaged the ligations with both the Packagene
extracs and the Stratagene Gigapack extracts.
The cloning of the 12kb fragment worked some months ago. Now there
is nothing, the results are (almost) nil. Does anyone have suggestions to
our problem? Could it be contaminants from the agarose?
Is it ok to use GeneClean instead of phenol extractions? What should we do?
I would appreciate some advice.
Bjoern Forbord
Odont Inst Physiol Biochem
University of Oslo
Norway
--
Bjoern Forbord
(bjoernf at karius.uio.no)
Odont Inst Physiol Biochem
Dental Faculty
PO Box 1052 Blinderm
N-0316 OSLO 3
Tel: +47 2 456029
