Fed up with my PCR

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ahbu
ahbu's picture
Fed up with my PCR

 I am using an un-optimized touchdown PCR to screen a couple of microsatellite primers designed by a commerical company. The results aren't looking good and are not consistent at all.

#1-
I got this smear which I thought could come from loading the negative after the last sample. Negative is the one which is in between 2 empty lanes.

#2-
So I added the negative first. And it seemed alright except for the primer dimer? 

#3-
So I assume all's well and tried it again without changing anything. Negative in the first lane followed by ladder. 

IS THIS A CONTAMINATION?! Looks awfully like one. I had one previously and got new reagents, took the pippettes apart, clean them and recalibrate. WHAT IS THE PROBLEM?

Biju
Biju's picture
 Hi

 Hi
Let us try to figure out the issue and solve the problem.
Could you  add details of nature of template DNA(human, mice, plant etc), expected product sizecalculated Tm, touch down protocol ,% Gc content and PCR setup(volume,conc of Mg2+ ions) of all the microsatellite primers you are using?
Acoording to me getting some amplification is far better than a clean gel without any thing to observe, though negative control product is a probem that could be sorted out.
Biju Joseph