My RT primers have suggested annealing temperature of 42 degree celsius. I tried normal PCR with this annealing temperature and it showed considerable bands. Now, while running it on Real Time PCR there is no amplification at this annealiing temp., also I even tried 50 degree celsius, and it showed amplification only at 50 cycles. To my curiosity the NTC showed amplication too.I took Beta Actin as reference, and it too showed amplification with a cp value of 28 avg.. which is supposed to be below 20.
Now I am confused with two thing here. First, is my annealing temp not correct.? Second, is my cDNA being degraded? Can anyone suggest me on this?
I think there are some variations between the predicted and real time annealing temperature. After I designed a primer, usually I was taught to optimize the PCR first by using different temperature. For example, if the predicted temperature is 54 degree celsius, I will try to run PCR at 5 different temperatures, which include 50, 52, 54, 56 and 58 degree celsius to see at which temperature could produce higher yields.
did you reduce your template concentration or used the same PCR concentration
How did you determine cDNA concentration ?
How much RNA did you use for each cDNA reaction and what is the final reaction volume ?
I doubt that your cDNA concentration is lower than you think.
Actually my RNA conc. was 2000 ng/ul which I diluted half and performed cDNA synthesis. SO accordingly the cDNA should be roughly of same conc. . I just checked the cDNA conc on Nano drop spectrophotometre which is not so certain. But I expect my cDNA to be around 1000 ng/ul, thats it.
Ok, which assay are you using and what is MgCl2 concentration in your qPCR reaction ?
high concentrations can cause a considerable increase in primer melting (and annealing) temperature.
another important thing, have you done a DNAse step before cDNA step and have you tried if NTC gave bonds with normal PCR ?
I am using Roche SYBR Green Master Mix for qPCR, so the MgCl2 conc. comes optimised. I didn't perform any DNase treatment before cDNA synthesis, haven't tried NTC with normal PCR.
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