SREBP2 primer seq is
Forward 5'-GGAGACCATGGAGACCCTCAC-3' (GC%-62, Tm-65.3) reaction conc 10pmol/ul
Reverse 3'-AGACAATGGGACCTGGCTGAA-5' (GC% 52, Tm-61.3) reaction conc 10pmol/ul
I used several annealing temperatures (53,55,57,60,62) however I did not find PCR product. The cDNA quality is fine. following reaction mix used for PCR
2x master mix-(ready for use)-----12.5ul
F primer---------------0.4ul
R Primer---------------0.4ul
cDNA--------------------2ul (1ug/ul mRNA used for cDNA synthesis)
H2O----------------------9.7ul
Following PCR condition is used
Initial denaturation------95-----for 3 min
Denaturation---------------95-----for 40 sec
Annealing---------------------(53,55,57,60,62)--for 1min
Extension--------------------72-------- for 40 sec
Final extension------------72--------for 5 min
Final hold------------------10
Could anyone suggest to me, how to calculate annealing temperature and what was the possible reason for my default PCR reaction?
Hello,
you can check your Tm using online tools like this
https://eurofinsgenomics.eu/en/ecom/tools/oligo-analysis/. Further, you can check primer for specificity and also Tm using https://www.ncbi.nlm.nih.gov/tools/primer-blast/.
However, Tm might not be the issue here. What tissue are you analyzing and are you sure, the SREBP2 is expressed?
Best
Soner
Hello Soner Öner-Sieben
According to the previous work, SREBP2 should express.
Hi Md Asrafuzzaman how are you doing today?
The Tm of the primer is the one we usually use as annealing temperature. The Tm is based on the GC% content in the primer. You tried different annealing temperatures but is bellow the temperature of your forward primer. Thus, your forward primer is not annealing during the PCR reaction.
Also, there is a 4°C difference between your forward and reverse primers, this is not good for any PCR reaction. Differences about 1 degree works sometimes, but it is not recommended for qPCR.
I suggest you design a new primer pair with a less difference temperature between them. Primer blast from NCBI platform is good for it. Choose primers with Tm around 59, 60 or 61°C.
Most of qPCR supermix usually operates in two steps PCR. The first step is the denaturation, while the annealing and the amplification occurs at the same temperature and is usually standardized at 60°C. However as describe in your question, you are working with a three step PCR reaction.
Try a new primer pair with Tm around 60. This should works fine and the PCR product does not need to be big, around 150bp is enough for qPCR.
Well, I will hope it help you.
Best regards.
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