I performed real-time PCR on cDNA of samples that had a nanodrop quantity of 1000+ ng/ul, but when i perform i do not get any proper results. i am attaching the report of my real-time. kindly help me out. should i synthesize the cDNA again or should i change something in the way i'm performing real-time pcr.
Before you go through all the work of remaking cDNA try a few tests. Try standard end-point PCR with your primers for your target and your housekeeping gene. Ask if anyone else is getting odd results with the machine in case it's a calibration error. How much cDNA are you using per reaction?
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when I had Put the pcr tubes in the thermocycler for cDNA synthesis, there was a power breakage of 5 minutes after which the reaction resumed. I performed the nanodrop which gave satisfactory results, the concentration and purity of the cDNA were both good. But after performing real-time pcr, I’m not getting results. should I synthesise the cDNA again too or should I use the previously synthesised cDNA (used in this reaction) for real-time again?
If the power went out DURING your cDNA synthesis, then the most likely explanation is that you didn't synthesis any/enough cDNA.
The nano drop will only tell you the concentration of nucleic acids - not the type of nucleotide, length of molecules etc.
Try again and good luck!
I agree with Katie A S Burnette. The nanodrop does not tell length/success of synthesis, just how many nucleotides are present.
Your housekeeping gene likely worked as those tend to be high copy number. You probably had enough of that template made to meet a threshold for amplification, while your target genes were likely less abundant in expression, so there wasn't enough to detect.
Make more cDNA and try again!
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