It might sound lame but I am new to it. I don't get first I must optimize the primer or check for my qpcr efficiency by serially diluting my cdna.
Can anyone briefly explain how I can do these things? or if there is a link I can follow.
I am using sybr green assay and applied biosystem step1.
You should check:
1) Primer dimer
2) Ampicon size
3) The best annealing temperature by running a thermal gradient (somewhat- optional).
Once you have done that, you can make a cDNA serial dilution, i.e. by 10 which covers 6 logs. For example you can make 1:10 dilution of your stock cDNA by taking 10 uL of stock cDNA + 90 uL H20, which can become your 1st unit, then repeat this dilution step 5 more times. So now you have 1:10, 1:100, 1:1000, etc. Ideally, perform in triplicates hence why we make 100 uL for cDNA. You can use the rest for other primers or re-runs.
A typical PCR may ask for 5 uL master mix, 4 ul cDNA and 1 uL primer (F+R). Now you just add your cDNA given that master mix is the same for all. Since we are doing 10X dilutions, we except to find sqrt(10)= 3.1 cycle difference. Most modern PCR machines have software that help you calculate the efficiency from your input.
Hi Fatima,
I hope it would be wise to check the primer oligomarization first. first you should check the primer product size, primer dimer and best anneling temperature.
Dear Sulail:
I strongly recomend yo the next manual:
https://www.agilent.com/cs/library/brochures/Brochure_Guide%20to%20QPCR_IN70200C.pdf
If you are new in the qPCR world you must read all the manual. If you want to know about primer optimization, skip to page 47.
Best regards!!!!!!!
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