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PCR don't work

Andre Hamel hamel at cc.umanitoba.ca
Sat Jul 2 10:08:51 EST 1994


I'm inclined to pose a few questions to YOU, in midst of offering any
ideas:

1/  I assume that this is a cDNA library that you're assaying?
If so, have you screened it in any way (other than by PCR)? ... such as by
immunoscreening or by DNA probing.


If cDNA library, was poly A+ RNA used (assuming if eukaryote origin). 

(RARE, but still possible:  perhaps there's heteronuclear mRNA that had
been cloned ... hence, your cDNA of interest may be significantly larger
than expected ... due to presence of introns ... thus would  *resemble*
cloning of genomic DNA of that RNA transcript).

If immunoscreened, consider that PURELY random chance of ANY one clone
being in correct orientation AND correct reading frame is 1/2 x 1/3 = 1/6.
Thus, only 1/6 of ANY particular cDNA species MIGHT be able to be immuno
screened ... that's assuming also that the immuno probe (poly- or
mono- clonal Ab) is capable of detecting the antigenic determinents AS
EXPRESSED by that system (in this case, MUST detect amino acid sequences,
since (dogma) E. coli  *doesn't*  glycosylate proteins in same manner as
eukaryotes). ... FURTHERMORE, if that particular protein/peptide turns out
to be toxic to E. coli, then NEGATIVE  *selective* pressure can result ...
in short, such cells might NOT survive and hence NOT appear to be
represented in the cDNA library ... thus, wise to NOT induce expression
(such as by inclusion of IPTG in agar) ... and consider alternative 
*screening*  method, such as by DNA probing (or, I suspect, by PCR of
sub-sets of the library pool) ... hope I haven't digressed to much so far.
 :^) ... yickity, yack ... the cat came back, the very next day ay ay o yay

2/ Was the cDNA in some way "force orientation" cloned? ... such as by
using specific cDNA synthesis primers containing RE site sequence(s)? If
so, perhaps then your particular cDNA of interest CANNOT be PCR amplified
using T7 and gene specific primer ... unless you PCR "all way 'roundd the
vector ... the "long way around, so to speak"? ... in short, that library
pool may not have any of your cDNA in correct orientation.

Have you considered using TWO genespecific primers (taking aside obvious
added cost? ... although time does = $$ ... then again, grad student time
is oft' considered cheap $$ ... AND, also good way to learn ... the "hard
way", etc.

3/ The cDNA of interest is toxic to E.coli (even if expression is NOT
intentionally induced ... there's ALWAYS some "leaky" expression ... even
in VERY tightly expression controlled systems (such as in E.coli over
producing Lac repressor) ... in your case it's T7 promoter ...

Does  *anybody*  in 'netland know how expression from T7 promoter can be
controlled?

best wishes,

                            ********************
Andre Hamel                                    email: hamel at cc.umanitoba.ca
Manitoba Government Veterinary Services          lab tel.: (204) 945-7630
545 University Crescent,                              FAX: (204) 945-8062 
Winnipeg, Manitoba, 
CANADA   R3T 5S6            ********************


In article <2us3ur$2d3 at mserv1.dl.ac.uk> "cilia jonas t2814" <Cilia.Jonas at vkl.uni-regensburg.de> writes:
>hello netters
>I try to make a PCR with a lambda-ZAP-Phagenpool
>One primer is T7, hybridizing to a sequence common to all phage DNA
>the other ones are genspecific (20-25 bp long). I can amplify with two 
>genspecific 
>primers a short stretch, so I know it is there in my phagepool.
>A PCR with another (strange) genspecific primer and the T7 worked 
>well, 
>too. But I fail  to amplify something between my genspecific primer
>and the T7
>I'm very grateful for any suggestings and ideas
>Sincerly  Cilia





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