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Home made real-time PCR problem


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#1 ecnuhh

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Posted 07 February 2012 - 07:14 AM

I have problem for my home-made real-time PCR. Please see attached pictures. Everything looks good, except the melting curve (figure 1). You can see at the low temp., there are very strong signals. I do not know what is that cause. I ran those products on gel, and it shows very clear band (figure 3). Does anybody have any comments? What is the reason to have those signal?

Many thanks.

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#2 bithorax

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Posted 07 February 2012 - 06:55 PM

It looks like your primers are not specific, so there is non-specific amplification.
You can try to optimize the reaction (rising temerature, changing concentration of primers) or just redisign primers or, better, try to find published one - they tend to work.
if you will not manage this a probe would be solution, instead of SYBR Green (or other dye you're using)

#3 ecnuhh

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Posted 08 February 2012 - 08:14 AM

Thank you for your advice, bithorax. Actually, the primers are published one. We used these primers for our published paper. When I use the SYBR Green premix from company, everything works (no such messy peak on melting curve). You know the reagents is expensive, so I want to make it. Till now, everything looks good except the melting curve. I do not think those peak is dimer. I definitely need to optimize the reaction. I need more suggestions.
Thank you again.

#4 bithorax

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Posted 09 February 2012 - 06:09 AM

so you are a brave guy to do it by yourself :)
I would try things like with normal PCR - increase Mg ions, maybe add DMSO - but this I dont know how would it work with fluorescent dyes.

I would like as well to turn your attention to Bioline, they have very good, cheap kits, for 500 reactions we pay $220. While hot start polymerase costs almost the same.

Anyway good luck!

#5 Curlis

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Posted 18 May 2012 - 10:33 AM

It looks like your primers are not specific, so there is non-specific amplification.
You can try to optimize the reaction (rising temerature, changing concentration of primers) or just redisign primers or, better, try to find published one - they tend to work.
if you will not manage this a probe would be solution, instead of SYBR Green (or other dye you're using)

Hi,

Can you please send me your recipee which you are using for making master mix, because even I am trying to get using only SYBR Green with the master mix which I prepared using Double distilled water, 10X Poly buffer, MgCl2, dNTP's, F and reverse primer and Taq Polymerase and SYBR Green. But till now no luck!!!!




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