Problems with SYBR green assay - (Dec/13/2007 )

I`m having trouble with a SYBR green assay for a 78 bp product on the LC480 machine. The curves reaches the plateau phase very early and at the same time they are kind of "wavy". I have with some success reduced the waves by changing the primerconcentration. I have run 10 fold dilutions, positive and negative controls. I am able to differentiate between positive and negative samples (my primary goal), but I am not satiesfied with the strange shapes of the curves. I use 5 ul template (DNA concentration ranging between 13 - 135 ng/ul). Originally the protocol is taken from a conventional PCR, and I have applied exactly the same program for the LC480. I have to admit that I'm quit new in this game and would be very grateful if anyone have ideas that could improve my run.

Thanks...

[attachment=3941:Amplific...n_curves.ppt]


I also ran the products on a 1.25 % agarose gel --> negative

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I'm afraid we are going to need more information to troubleshoot this: which program setup are you using, primer concentrations, etc...
What I can see is that first your template is amplifying far too early, so I would try diluting it further. Also, is not generally a good idea to translate a conventional PCR program into qPCR, as in qPCR you are going to use far shorter cycles...

If you provide us with some more information we might be able to help you...

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Thank you for your answer. Here is some extra information.

This is the temperature program I used:

94 C- 5 min

94 C - 30 s
48 C - 30 s
72 C - 30 s

72 C - 5 min


Primerconcentration: 0.25 uM
I have also used the LightCycler 480 SYBR Green I master kit.

Dilutions of all samples were made ( 1:10 ----> 1:100 000) with no success (only non diluted and 1:10 dilutions seem to be "positive")...Maybe I could try to change the time settings in the amplification step or is it possible at all to make this function with the RT-PCR machine?

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The weird thing is that you have no products visible on agarose. The PCR reaction should lend somthing visible, as a plateau phase is reached. So, maybe the amplification you see is not the real product, but another thing (like primer dimers, etc). Every time you have weird amplification curves reflect a problem with your amplification. I suggest you try different primers.

Another question : have you ever got a good amplification with other primers on this machine?

And as for the program, it seems fine to me. And I also use 250 ng of primers. Hope this helps!

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Sfinx:
Can you post the raw fluorescent data too? (Experiment - Data tab - Fluorescent History graph)

On the first look it seems that amplification has a nonexistent baseline, that is usually caused by high concentration of sample, but hard to tell without raw data.

Then, your annealing temperature is quite low, usually primers are designed specially for real-time so that they work at 60 °C annealing.

Just an idea.. standard amplification program for SYBR on LC480 is:
95 - 5min
cycle
95 - 10 s
anneal - 10 s
72 - 10s

That should be enough for such a small amplicon anyway. Try that.

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