Yet more PCR problems

Shahram Mori smori at nmsu.edu
Sun Jan 29 16:32:59 EST 1995


LMS  (Tel 0635 578411 X2598) (lee.smith at afrc.ac.uk) wrote:
: Heeellllppppppp!!!!!!
: Here's the story so far:
:  In the course of developing my PCR reactions,
: I designed one upstream and three downstream primers.
: For no particular reason, I decided to use the middle
: of these three primers in my 'standard' PCR test.
: Life was going swimmingly, all was right with the world.
: I used the system on DNA samples from tissue culture system,
: from blood samples, from tumour samples, nothing could stop
: me.
: UNTIL: just before christmas, I ran a titration of target,
: and all I got were smears. I repeated it (twice), I got smears.
: I changed all my reagents, smears. I changed my PCR machine, smears.
: I went back to the beginning, and used my upstream primer with the 
: other two downstream primers- worked perfectly. A Ha I cried, my
: primer has gone funny. I ordered more primers. They arrived.
: I used them. Smears again (and sometimes, absolutely nothing).
: I have almost decided to switch the whole test system to one of 
: the other downstream primers - this, however, means repeating a whole
: lot of work (about 18months worth, including data for a paper
: and seminar that I've just written).
:  Does anybody know what is happening? Why did my PCR suddenly go wrong
: after two years? Why didn't buying new primers solve the problem?
: Lee Smith
: IAH Compton
: P.S. I blame the government

Could it be that your DNA has fragmented? It might be that there is a
contaminating piece of DNA [ a fragment of the original] where the primer
binds to and that gives you smear. I would recommend running your DNA on
agarose to see if there are any other contaminating DNA's there. This is just
an idea, probably a long shot.
Cheers
--
Shahram Mori					   _/\_
Program in Molecular Biology			  _\  /_ Saskatoon/SK/CANADA
Dept. of chemistry and Biochemistry Box 3C	  \_  _/
NMSU  Las Cruces NM				    ||
88003





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