Melting Curve Analysis Difficulties - (Feb/13/2009 )

So I'm having obscene difficulties getting a real-time PCR to work and figured this could be the place to help.

I'm currently trying to amplify a CpG island region, and for some reason, my DNA will not work unless it has been bisulfite-treated, a process that takes me two more days AFTER I have salt-precipitated the DNA from the cells. I'm working with SYBR Green, and the fluorescence starts up abnormally high from the very first cycle, then drops as the PCR continues and rises a little bit at the very end. The melting curve, which I desperately need to be clear, ends up looking like a drunken sine function with squiggles and little bits all over rather than a nice clean peak.

The Steps:
12 min at 95 deg C to activate the Hot Start
Cycling
1 min at 94 deg C
1 min at 66 deg C (Increased to avoid getting primer dimers)
1 min at 72 deg C
40 to 50 x

Melting Curve
1 min at 95 deg C
5 min at 60 deg C
Melting curve from 60 to 95 over 30 minutes

I've tried altering the annealing temperature a bit, increasing the annealing time, and checking the concentrations of the primers. But nothing has really changed. I'm considering adding some DMSO as a PCR adjunct, but I'm not really sure if it could improve something that isn't amplifying already (that I can tell)?

Does anyone have any advice or tips that I could use to get this to work properly?
Thanks for any help,

Bryan

Hai,

Whats the size of ur amplicon and wahts the initial concn of DNA u use?

BAZorko on Feb 13 2009, 09:42 PM said:

My current amplicons are about 98 bp and 108 bp. I haven't been able to calculate the concentration of DNA very well since the UV spec has been being wonky lately, but I use 3 ul in a 25 ul reaction. The last ones I was able to calculate were averaging about 40 ug/ml before I but them in the tube. So...calculating that out gives 0.040 ug/ ul, then 0.120 ug in 3 ul. 0.120 ug/25 ul give about 0.0048 ug/ul.

I'm not sure if that helps.
So a recap if you couldn't follow my rambling
Amplicons are 98-108 bp long
is not precise, but generally around 0.0048 ug/ul in the reaction vessel.

Not sure what the Tm of your primers are, but the high annealing temp may be an issue, which might explain the late rise. If the primers are designed so that they do not interact (esp. @ the 3' ends) then a lower Tm should be ok. Also, what sort of salt concentration is your input DNA in? If there is too much salt, it could also adversely affect the melting temp. of the oligos.

High initial fluorescence may be a sign of too much template in reaction, did you try to make dillution series?

I use a high-salt concentration to precipitate the DNA, but I transfer the DNA once precipitated to a 1x TE buffer at pH 8.0. The concentration of the lysis buffer I use to extract the DNA from the cells has about 400 mM salt concentration. The TE is 10 mM Tris-Cl and 1 mM EDTA. We got the primer design from other papers, and they've been referenced and repeated in a bunch of papers. For some reason, in our lab, we were getting a lot of reactions in the negative control while doing this, so we increased the temperature to 66 from 64 for the annealing. It's a pretty high GC content region, but the primers are put to about 50% GC to help that.

Thanks for the idea about the concentration gradient. I'm going to give that a try today with a 1x, 0.5x 0.25x and 0.125x concentration of the DNA from the tube.

On second thought, with the levels of fluorescence I'm getting, I think I'll try 1, 0.5, 0.1, and 0.01.

Result! I managed to get some reactions with the lower concentrations of DNA. Thanks!