Dear friends!
I designed, checked, and ordered primers for RT PCR. How can I verify them as working now?
1. How to choose the temperature for best amplification (the primers Tm is 60, recommendation for my hot-start polymerase is 60-65 degrees annealing)?
2. How to choose an optimal concentration of primers? Are there any approximately mean values?
P. S. I have limited access to the PCR lab so my experiments have to be "predesigned" as best as possible.
I would be glad to hear all your considerations. Thx in advance)
Have a nice weekend!
1. If you have a classical PCR machine, run a temperature gradient +/-10 degrees vs Tm
2. 200-400 nM is generally a good start, run the PCR product on a 2% agarose gel - if you see primer bands, dimers, it means the concentration is too high
Hi,
1) I would suggest to do gradient run for the primers, according to the temperature you mentioned i.e. 60, I would do the gradient run from 58 to 64 C.
2) For the qPCR run, the concentration of the primer can be decided based on the titration from 100nM to 500 nM as the final concentration of the primer in the test. Based on my experience, I always make 100uM stock of the primers once received and then make 10uM working stock. From the working stock i use 1uL in 20uL test solution. It always gave me best results. run the sample on the agarose gel to see if there is a single band and also will give you an idea which temperature(Tm) was the best based on the intensity of the bands.
3) Run a melt curve in your qPCR run just to get an idea of your sample quality and primer specificity. Multiple bands represents contamination in your sample and also primer specificity is not good.
Hope this helps!
Good luck!
Anna Zhukovska
1.Ta for 58-60 C should be ok.
2. Primer concentration: check the info sheet coming from your PCR kit. Usually, they have a suggested concentration.
3. Make sure your primers are from exons of the gene, not introns, since you are doing RT-PCR from mRNA.
Péter Gyarmati what do you mean by running a temperature gradient +/-10 degrees vs Tm? Can you please explain in detail?
Darshini Desai
1. It`s a good idea, but I am really limited for doing gradient run from 58 to 64 C(
2. My suggestion is to do a primers titration during one PCR experiment.
3. Yep, melt curve will be done absolutely)
Yuan-Yeu Yau the manufacturer`s recommendation is to use 100-200 nM.
But! For economy, we usually use a 2 times smaller volume of EvaGreen supermix (it works OK). Should I use 300-400 nM of primers then? Am puzzled with calculations...
Anna Zhukovska
I would still use their suggested amount: 100-200 nM. If you want to see 300-400 nM's results, you can add 2 PCR tubes with such concentration.
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