Protocol Online logo
Top : New Forum Archives (2009-): : PCR, RT-PCR and Real-Time PCR

Interpreting melting curve data in Sybr Green RT-PCR - (Nov/06/2009 )

Pages: Previous 1 2 

hi pop i saw the curves... i striongly feel that the amplification protocol needs to be worked on.. although tere may be different opinions too!!! 58 degrees seems to me too low a temperature for annealing given the GC content in your primers which may be the cause of many melt curves due to non-specific binding.. did u run a gel or this pcr product ever??!! do you get multiple species tere too?? in that case i suggest do a gradient for primer annealing and try running a gel till a single bnand of your interest and then u can switch back to real time!!
why i say 1X is that along with the sybr green, there are dNTP,s taqm etc present.. may be the 0.5X sybr green s not making a difference but the otehr components can!!!

-Pradeep Iyer-

Thanks Pradeep. Yes I did a regular PCR (temp gradient 55, 58 and 60C). My results are:

1. At all temperatures, the positive samples amplified a single band at the same intensity. The band is <200 bp but >150bp. Is this ok?
So I intend to check annealing temp to higher than 60 in the regular PCR, then optimize in the real time PCR machine later.

2. I got very faint fuzzy band in the negative controls at all temperatures tested in regular PCR. The band size is less than my product above, but it is very close to 150 bp. I don't have these bands in my positive controls (unless the product because of its high concentration "masks" the fluorescence of this non-specific amplicon? is that possible?). primer dimers are only around 100 bp or less right?

I'll let you know at the end of hte day what I get for my regular PCR temp gradient analysis.


Sorry for this delayed update. I ran a regular PCR at higher temps (58 62 66 70), and I got amplification only up to 62 (band is less intense of course than 58. Should I still try gradient between 58 and 62? what else should I consider when I start optimizing in real time machine? thanks again!

Pages: Previous 1 2