Could SYBR GREEN I see 60bp dsDNA? - (Apr/05/2008 )
My target is only 60bp dsDNA. I ran QPCR yesterday. There is even no exponential state!
It's heart breaking.
SYBR GREEN I could bind to 60bp dsDNA. Is it a sure thing? (Bio-Rad IQ supermix SYBR GREEN I kit)
I set up my experiment in a PCR hood without turning on the light in it. The whole set up time is around one hour. Is it possible that the SYBR Green I is already deactivated by light?
Guess I have to find a dark room to set up my experiment. Sigh~
Thanks a lot, guys.
Good luck to everyone!
Hao
I'm using SYBR Green I mix (Roche LC480), never cared about light while setting the experiment and my products are often 60-70bp. And getting nice curves.
What actually do you get at the end? No signal, low-signal, high- linear/nonlinear curve? What about melting curve analysis, how does it look? It's your first SYBR experiment, what is the cycle and where in it you read the fluorescence? Did those primers ever worked before or it's a first test?
More info needed.
No fluorescent signal, every curve is flat. Even the melting curve is flat.
I've tried it three times. All doesn't work.
Interestingly, after the melting curve analysis, I run a gel using the stuff in the same QPCR tube, I could see the desired band clearly without any contamination, nor primer dimer.
I guess something is wrong with the fluorescent dye.
I guess the SYBR Green was dead during experiment setup.
I now know that if we want to do some science, we should have a strong heart in the first place.
Please have a look at the gel in the attachment.
Thanks anyway.
Hao
What actually do you get at the end? No signal, low-signal, high- linear/nonlinear curve? What about melting curve analysis, how does it look? It's your first SYBR experiment, what is the cycle and where in it you read the fluorescence? Did those primers ever worked before or it's a first test?
More info needed.
Nice bands I usualy get more dimers 
Yes, everything points out that your SYBR mix is completely spoiled. Is it a new one, or one stored for some time?
Or the machine could be broken. Have you tried a known working reaction recently?
A very basic question, but still important : are you sure that your machine is calibrated to read the syber green?
Yes, I will check that.
Thanks.
I've email Biorad, they hasn't replied yet.
They've used this machine getting pretty good SYBR Green result.
Sigh~
It's not ours. It belongs to some other group.
Ok, very stupid question, but where in your protocol is the adquisition of fluorescence placed?