Originally Posted by butters
Marco, if you still read this.
We will never know until you have get the primer pair and test it out. For me, 200++ (just in case if ppl are thinking over 300 or 400 or 500++ bp, no I don't meant that high) is still good to try if really no other option available.
Since you mentioned bimodal peak curve, I would have to think that your rt PCR machine can do melt curve. That would help but it is not definitive. But it is a guide. For example in your "interpretation of result: you need to state the range value of your said product is positive and everything else is negative".
Bimodal or anything more than 2 peak would be attribute to formation of primer dimer. Unless your primer pair is really that BAD, if not you could still do some optimization. Do consult your rt pcr manual for steps on optimization.
Are you making a primer pair with both unique seq or one is unique follow by another not so unique?
Yeah, I'm still reading, and I will at least until I have them and tell you guys how it all worked out.
I've read in the last week that it's very good to check the PCR product with melting curves and electrophoresis, and I plan to do so. In that way, I could see if the bimodal curve is a result of dimerization or just heterogeneity to melt. And I hope my primers won't be so bad, they have some nice DG values for either hairpin, homodimer and heterodimer... so I really hope that won't be a problem. I will consult the manual, though.
Originally Posted by namishu95
SYBR Green usually works with amplicon between 100-150 bp, the data that SYBR green assay generated by over 200 bp amplicon is useless.
I donít think the PCR sensitivity can be improved with extending the polymerization time. it mainly depends on primer-probe design and secondary structure.
I'm sorry, I'm a little slow and I want to be sure that I'm understanding. Do you mean the data will be useless as if I shouldn't even try it out?
I'm not really into the quantification, although it would be nice. I kinda only need to know if there was any amplification or if there wasn't... don't you think it could work?
I really appreciate the help of both of you, I just wanna be sure that I'm really getting what you say.