SYBR green: melt curve Tm shift

[Ed Siefker]

I am having a small problem with my SYBR green qrtpcr.
I have 4 primer sets running against 2 different cDNA
samples. The primers are all at the same concentration
(300 nm). I prepared both cDNA samples side by side, in
exactly the same way and loaded 10ng of each.

The problem is, when I look at the melt curve, I'm seeing
a decrease in Tm of the product of about 1 degree C from
one cDNA to another. This Tm shift occurs for all 4 primer
sets. In both cases, there is a single sharp peak, so I'm
doubting I have primer dimers.

Does anyone have any ideas? I've used these primers before
on different samples, and the melting curves always exactly
coincided, no matter what the cDNA was.

[Duncan Clark]

Historians believe that in newspost
<mailman.11.126142...@net.bio.net> on Mon, 21 Dec 2009,
Ed Siefker <ebs1...@creighton.edu> penned the following literary
masterpiece:

>
>Does anyone have any ideas? I've used these primers before
>on different samples, and the melting curves always exactly
>coincided, no matter what the cDNA was.

Something in one cDNA sample that isn't in the other. Could be salt, Mg,
EDTA, glycerol etc. It doesn't take much to shift 1C.

I bet if you could desalt it somehow (G25 or G50 spin column), it would
be fine.

Duncan
--
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.