Dear all,
I am facing a problem with my real time PCR experiment. I had isolated RNA from a strain of interest (RNA conc. 25 ng/uL) and carried out cDNA synthesis with 8uL of the RNA sample (final CDNA reaction volume 20 uL). Following cDNA synthesis, I carried out Real time in a final volume of 10uL reaction for 45 cycles. The reaction did not generate CP values although I could see bright bands in the gene corresponding to my gene of interest. I used SyBr green dye 2X (volume 2uL) in the reaction mixture. Is this due to only problem of template conc or any other factor might be contributing. It will be of great help if you could answer the same.
The syber green is 2x of the working concentration. So in 10 ul of reaction u have to add 5 ul of 2x syber green.
Hello,
You should take 300-500 ng mRNA for cDNA synthesis. Before cDNA preparation you have to checked the 260/280 as well as 260/230 value > 1.9-2. When you are using qPCR reaction always dilute your cDNA 1:10 for good result.
Better you follow the manual of the particular SYBR green master mix which one you have used.
Hope it helps you.
Hello,
if you don't have Cp value for your housekeeping gene in qPCR, maybe the problem comes from cDNA synthesis ? Or, did you check your primers? Do you have positive control?
Your Sybr green volume may seems low to me (you may use 10ul) also re-confirm the concentration of your standard and the calculation of standard template used if you are very sure of your reverse transcription outcome.
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