I would like to determine the best annealing temperature for my primers, giving the lowest Cq and the best efficiency. Is there a thermal gradient feature on the Roche LC480 instrument? And if so, how should a plate look like?
Hi,
Were the primers designed by you? Or taken from literature? In both condition, their annealing temperatures should be 60C degree as optimal. I suggest you to just run a conventional pcr and check the products on gel. Later, you should calculate primer efficiency by serial dilution reaction on qPCR.
You do not need gradient qPCR experiment. Do not wate your mastermix. Just google qpcr primer efficiency.
Good luck.
Cheers!
yes, there is a thermal gradient feature on that awesome machine. It is vertical (column-wise)and you can select temperature difference up to 20 degrees in the range of 37-98 degrees. The plate should be designed accordingly that is you have to check that which wells will provide what temperate when you have selected gradient.
please feel free to ping
- Yes there will be
- You need to perform this function to check primer efficiency > 90% and ideally > 95% such that relative gene expression calculated by Livak or Delta Delta Ct method pr supposes a 'perfect doubling' of 2 or in line with what I have said 0.95 x 2.0 on average per cycle
- To do this you will set up triplicates of the same biological replicate, i.e. technical x 3 for your neat cDNA then perform a 1:2 serial dilution x 8. Make sure you vortex each dilution before using to make the next
- I say a 1: 2 serial dilution: But that pre supposes that your gene copy number is such that your expected Ct is 25-30cycles
- If your gene is say structural reminiscent therefore of a housekeeping gene you perform a 1: 10 log dilution x 8
- You need to do this for every primer pair including your reference genes
- It is OK to perform different dilutions for your housekeepers = high copy number (1:10) and actual GOI if low copy number (1:2 serial dilution) but make sure you use the same cDNA (biological replicate) for each
- Furthermore, if you are testing 2 different conditions, i.e. treated versus untreated, making a pool of the 2 cDNAs for the purposes of this standard curve
- Having found the Riohe software and set up the plate as decribed make sure your resulting primers yield R^2 > 0.98 and efficiency > 90%
- If you found that your last two point from a log dilution result in spread of your triplicates such that they differ by > 0.5cyles from the mean, then it is OK to remove them
- As a minimum you need 6 dilution points (although 8 better)
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