Protocol for qPCR using the ABI SYBRŪ Green PCR Master Mix - (Sep/11/2013 )
does any one of you have experience working with the Universal 2X Sybr-Green PCR mix from Applied Biosystems (Product No. 4309155) and could suggest a nice qPCR protocol regarding:
1) the total reaction volume
2) the final primer concentration in the reaction
3) the amount of cDNA template
4) the amount of the master mix
5) the thermal cycler program
I have used SYBR Green. I usually used a total reaction volume of 12uL. The final primer concentration would have been 2-10uM (I played around with this and the results stayed consistent - trying to determine the minimal amount of primer I could use - check SYBR green protocol). I would add 0.5uL of a 2ug cDNAtemplate. I would add 6uL of the SYBR Green master mix.
My thermal cycling program:
Doublets of the reaction mixture were loaded and cycled once at 50ºC (2mins), and 95ºC (10mins); 40 cycles at 95ºC (15secs), 58ºC (30secs), and 72ºC (30secs); and a final cycle at 95ºC (15secs), 60ºC (20secs), 95ºC (15secs), and 60ºC (15secs).
The 58C will be unique to your primer Tm's. I would always try to design your primer to a specific Tm. This will allow you to perform very diverse RT-PCR's simultaneously (saves plates and optical film).