Dear all,
I am trying to determine the efficiency of a set of primers for qPCR.
Being my very first qPCR... it doesn't seem too good to me. Please, seeing the image attached, does anyone have an idea of what I am doing wrong?
Should I increment the number of cycles? increase the dilution of my DNA sample?... check if I made a mess in the software settings?
Thanks in advance
Your PCR starts too late, try to increase the number of cycles (suggest 50-60) and/or increase the starting amount of DNA (make dilutions e.g. 200ng, 100ng, 10ng, 1ng, 0.1ng). See if this helps. Also try same amount of DNA with other primers you are aware of (worked before, taken from a trusted paper, etc.). If they work well with the current DNA amount, try to play with the annealing temp (add the annealing temp if it's not in the program) or design other primers.
You have low reproducibility of PCR-results and possible low analitical sensitivity. Firstly you should do QPCR optimization (any standard PCR optimization protocol for Mg and Tm - GOOGLE!). Then you should take some ten-fold dilutions (10-100-1000-10000) of your standard DNA matrix for each PCR reaction and do QPCR with calculation of the PCR-efficiency.
Hello
It is really to late. I think your thermal cycling is not adequate.
What is your qPCR type is it one step or 2 step?
IF one step , your reverse transcription is not adequate you should include more time ( more than 30 minutes) at the beginning for reverse transcription and hot start activation of polymerase for 10 minutes at 95 degree.
this may help
Did you try running your samples out on a gel? The amplification is so late that I suspect you may be seeing primer dimers. If the product sizes are what you expect, then you need to start with more template DNA. Right now, you should start with running out your product on a gel. You may need to take a step back and test out your primers using regular PCR just to make sure they will amplify your product(s).
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