PCR rxns give smears. Any advice?

Jeremy Weinman jjw at rsbs0.anu.edu.au
Sun Nov 29 20:37:42 EST 1992


Hi there nettlanders,
	we've been carrying out a series of PCR rxns from cDNA and up to
2 weeks ago everything was working OK.  However, now we find that we don't get
nice discrete bands anymore but get instead a smear from many kb down to tiny 
in size.  We are using a touchdown program 60-52dC in 0.5dC increments then 25 
cycles at 52dC.  We need this low temp as we'd used a T3+T(17) primer for 
the cDNA synthesis, and then T3 and an internal degenerate primer for the PCR, 
and the standard T3 primer is a pretty lousy one [we have a better one on 
order].  Previously we could go three succesive repeats of the PCR, using first
cDNA, then primary PCR, then secondary PCR as the target DNA - and get good 
clear products without any clean up between steps.  Now we only get a lousy
smear from the cDNA, and either nothing (!) or a horrible smear from amplified 
fragments we'd cut out and isolated.  Has anyone out there had this happen 
to them?  Or knows what might be going on?  Or can recommend a good reference
fro troubleshooting this sort of thing?

Thanks in advance for any help,
 
=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=
Dr. Jeremy Weinman				Email:	jjw at rsbs1.anu.edu.au	
Plant Microbe Interaction Group			Phone:	61 6 2495051
Research School of Biological Sciences		Fax:	61 6 2490754
Australian National University			Snail:	PO Box 475, Canberra, 
							ACT 2601, AUSTRALIA



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