Improving melt curves - (Sep/06/2008 )

Hi,

I am trying optimize a new qPCR experiment, but my melt curves are not looking very good and I would like to get some advice on how to improve them.

I have attached a melt curve and the details of my run are below.

I am using a rotor-gene 2000.

SYBR green master mix (Applied bioscience)
50 nM Forward and Reverse Primers
in 12 ul total

3 ul of cDNA reverse transcribed from 100 ng of RNA

in dilutions of 1:5,1:25,1:125,1:625

And the cycle conditions are

95 C hold 5 min
95 C for 30 secs
60 C for 15 secs
72 C for 15 secs

Should I be worried about the poor looking curve at the lower temperatures?
What is causing the high fluorescence readings at low temperatures?
Thanks, Brett

---

What steps have you taken to optimize your reaction? It looks like some wells have too much template, or the reaction anneal temp is too low, but it since some of the wells look good, my opinion is the former.

---

Thanks for your advice Randoramma.

So far I have just been increasing the annealing temp by 2 C steps, but continue to get similar results. I will have a go at lowering the template concentration and see if this improves it.

Thanks,
Brett

---

I'm not sure how lowering the Ta will help. Keep increasing the Ta until it starts to affect the efficiency of the reaction. If all else fails, set the temperature where you aquire fluorescence to be higher than all the cruft you are getting (which isn't too bad btw).

---

Hi,

I tried using a series of different concentrations. It seems that by doing this I did not reduce the cruft at the start, but my replicates seem to be quite nice and the standard curve is also good. I have attached the files of each of these.

Does this suggest that even thought there is this cruft at the start further optimization is not needed?

Thanks for any advice,
Brett

---

Your meltcurves an std curves look good. It indicates that you have 1 specific product in all replica's, the first part is most of the time pimer dimers in different lengths and forms (same length, but different Tm)

I use a rotorgene 6000 and have simular results with an very good optimised assay, (efficinecy = 104% GOI and 99% Internal control)

So I wouldn't worry about it

---