Riboprobes

vilimf01 at mcrcr6.med.nyu.edu vilimf01 at mcrcr6.med.nyu.edu
Sun Dec 8 15:19:20 EST 1996


Although I have never experienced this myself, I have heard some of my
friends noticed the same thing with some of their inserts.  One
potential explaination is that, since you are probably wsing a
blue/white vector, the fusion of your sequence to the lacZ is causing
the bacteria to become very unhappy.  This is especially true if you
are using DH5alpha's or some other non lacIq containing bacterial
strain.  In these strains, there is substantial leakage of lac
expression (i.e. the inhibition is poor).  It's easy enough to test,
just transform into XL1-blue or other lacIq strains.  I can say that
for some of my constructs, I have been unable to grow them in
DH5alpha's but was able to grow them in xl1-blue's for this very
reason.  Just a thought.

				Regards,		SVEN



In article <rjGSHAAvAoqyEwn7 at clorelab.demon.co.uk>, Clore Laboratory <CLORELAB at clorelab.demon.co.uk> writes:
> Dear netter
>         I have had an odd experience with my riboprobe synthesis. I hope
> someone from the scientific community will comment on this. I have been
> preparing riboprobes from at least 6 different sequences (cDNAs), by
> using the following procedure; I either obtain dscDNA (400-600 bp) by
> PCR amplification of vector inserts or from cDNA libraries. The PCR
> fragments are then ligated into a pCR-TRAP system (blunt-end ligation).
> Here, I expect that the orientation of the ligated inserts is random (is
> this correct). Then I reamplify the fragments, but now using primers
> that contain either the T7 core element for the T7 pol. or the SP6 core
> element for the SP6 pol.. The resulting templates are then used to
> synthesize either the T7 cRNA strand or the SP6 strand. This is a
> conventional procedure and the whole method works perfectly well,
> leading to high yield of cRNA. Naturally, you would expect the antisense
> to be 50% T7 and 50% SP6 derived, however 6 different cRNAs (6 T7 cRNA
> and 6 SP6 cRNA) have been produced so far and only the SP6 cRNAs are
> antisense to their cognate mRNA. Is this a coincidence or is there some
> mechanistic explanation for this.  Can someone comment.
> 
> Can anyone comment on this
> Regards
> V. Emilsson
> -- 
> Clore Laboratory



More information about the Methods mailing list