Problem on transformation

Bernard P. Murray, PhD bpmurray*STUFFER* at socrates.ucsf.edu
Sat Nov 21 16:07:05 EST 1998


In article <3656496E.6254BF08 at hotmail.com>, John Lum <johshi at hotmail.com> wrote:

> Hi,
> 
> Bernard P. Murray, PhD wrote:

What?!  I never did!

>     Talking about this rearrangement incident, I've recently cloned the
> promotor region of a gene. After transforming back to XL-1 Blue, I got clones
> of various length and even with different arrangement of restriction sites.
> Is it related to the rearrangement incident..?? I haven't got the sequence
> yet so I dunno if there are any inverted repeat or strange sequence in it.
> 
>     One of my friend suggest me to switch the host to SRB...do anyone got any
> information about it..??

You can check the genotype on Stratagene's web page;
      http://www.stratagene.com/tech_ref/genotype/genotype2.htm

e14-(McrA-) delta(mcrCB-hsdSMR-mrr)171 sbcC recJ uvrC umuC::Tn5 (Kanr)
supE44 lac gyrA96 relA1 thi-1 endA1 [F¹ proAB lacIqZ deltaM15]

This looks superficially like Stratagene's own SURE.
     I have never used SRB but I have used SURE (but never in a
direct comparison with a conventional strain).

I also had problems subcloning a promoter in pBluescript and
this appeared to be related to the toxicity as I could only
find it in one orientation (the wrong one).  When I moved
from pBluescript KS to SK, so that the insert would be in
the opposite orientation to Lac I had success.  When I had
quizzed this newsgroup on the topic two suggestions that
looked very promising were to delete LacP from the plasmid
or to insert a transcription terminator upstream of the
promoter (between LacP and the insert).
     ....and no, a lacIq strain did not help.

     Bernard
[no affiliation with Stratagene]
-- 
Bernard P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA



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