QPCR annealing temp help please!?

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Abhijeet Singh

why not just check the Tm of your primers

https://www.thermofisher.com/se/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/tm-calculator.html

Sharmin Suraiya

Hello Carrie Oh,

You can conduct your q-pcr at 60 ⁰ C annealing temperature.

For better q-pcr result you can use following software for primer design.

Primer3 (http://biotools.umassmed.edu/bioapps/primer3_www.cgi)

I am attaching a file here.

Sibnarayan Datta

You can try reducing the Tm by removing some 5' bases from the FW primer. Alternatively you can run a gradient pcr with this primer pair and find an optimum condition for this pair. Then you can use this temperature as the annealing+extension step in your qPCR run. Upto 65 degree is ok for run.

Maxim Yu. Sinitsky

Dear Carrie Oh,

What Master Mix do you use in your experiments? Commonly, the manufacturers give information about annealing temperature in their protocols. For example, I use PowerUp SYBR Green Master Mix, and I use 60⁰C annealing temperature for all primers that have Tm over 60⁰C (according to protocol).

Best regards,

Maxim

Carrie Oh

Hello! Thanks for your answers!

I'm using SYBR green master mix. I've used 60 Celsius for all my other primers however for this particular primer it has more nucleotides on fw than rv (28 vs 20) and the Tm is different for both. It mentioned on the thermo Fisher site that when over 20 nucleotides to increase by 3 degrees in the lowest primer Tm. Would increasing temp actually help?

Best wishes