Methylation of Plasmid !!!

mac-biocomputing2user user at mac-biocomputing2.EMBL-Heidelberg.DE
Tue Dec 17 04:40:06 EST 1996


Hi Folx.....




        ....I perfectly transformed XL1-Blue...with a plasmid...and to
make me sure that what I got from a Midi Prep (Quiagen)...was the
original Input I made some restrictions....every restriction sites I
checked corresponded exactly to the sequence I downloaded from the
web.....EXEPT for the Cla I sites !!!!
         
        There is a Cla I site in the polylinker of my palsmid, and in
theory (from the sequence I downloaded) there should be three Cla I in
my insert........in pratice by Cla I digestion I can only linearize my
plasmid ! I tried two different Cla I enzymes (NEB an Boehringer) + an
Isoschizomer Bsu 15 I and they all gave me the same result ! The Plasmid
linearized !
        I also tought the Cla I sites could be Methyleted in my
host.....and all of the three Cla I sites in my insert are actually
potential methylation sites by Dam Methylas (GATC....recognition
site....seen in NEB Catalog ). So I transformed back my plasmid in a
Dam- Dcm- Host (Stratagene SCS110) ....but today I had ONLY ONE
INSIGNIFICANT COLONY !(that I still have not checked). While my positive
control (pGEM series) didn't at all transformed SCS110 ! 

	Is there peraps any problem on transforming efficency when   methylated
plasmid DNA is transferred on a Dam- Dcm- Host ?


Please help me if you have any clue ! REPLY TO
corona at embl-heidelberg.de........many thanks!



------------------------------------------------------------------------------
DAVIDE CORONA   
EMBL - European Molecular Biology Laboratory
Gene Expression Programme 
Meyerhofstrasse 1
Postfach 10.2209
D-69012 Heidelberg
GERMANY
Phone:  +49 6221 356207 (home)
Phone:  +49 6221 387389 - 387490 (work)
Fax:    +49 6221 387518
E-mail: corona at EMBL-Heidelberg.DE
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