3 replies to this topic

[ecnuhh]

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.

Attached File(s)

•  1.pdf (46.88K)
  Number of downloads: 12
•  2.pdf (70.77K)
  Number of downloads: 8
•  3.jpg (7.7K)
  Number of downloads: 10

[bithorax]

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)

[ecnuhh]

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.

[bithorax]

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!