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Posted
ashu2007, on 16 May 2012 - 01:41 PM, said:
Hi,
I am new in qRT PCR. I ran around 5 times to troubleshoot my qRT PCR result. In mixture I am using double distilled autoclaved water and using SYBR green for this.
In disassociation curve I am getting multiple peaks. I know it can be because of primer dimer. But I am getting desired length of product which is required when I ran sample on gel after qRT-PCR. It just one band I am able to see when running qRT PCR product on gel. I am using 50Microlitre of reaction mixture for one well. I am using dliuted SYBR Green (10,000X) to 1X, .01X, .05X, 0.5X. I thought might be SYBR green is an issue, therefore I diluted it more.
I am unable to figure it out, can you please help me.
I am new in qRT PCR. I ran around 5 times to troubleshoot my qRT PCR result. In mixture I am using double distilled autoclaved water and using SYBR green for this.
In disassociation curve I am getting multiple peaks. I know it can be because of primer dimer. But I am getting desired length of product which is required when I ran sample on gel after qRT-PCR. It just one band I am able to see when running qRT PCR product on gel. I am using 50Microlitre of reaction mixture for one well. I am using dliuted SYBR Green (10,000X) to 1X, .01X, .05X, 0.5X. I thought might be SYBR green is an issue, therefore I diluted it more.
I am unable to figure it out, can you please help me.
I've had situations like this. Sometimes you'll see just one band on the gel but get multiple peaks in the melting curve. This could be because there are actually two peaks on the gel but they are too close together (running your gel longer may help) or there could be faint secondary products that you can't see on the gel, as the realtime PCR melt curve is a lot more sensitive that an agarose gel. Unless both my gel and melt curve indicate one clear product I don't trust the results and I either optimize the primers or design new ones. You could try an annealing temperature gradient and see how that affects what the melt curve and gel look like.
