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[DOKTOR MED]

Dear colleagues,

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

Kind regards,

Aris 

Edited by DOKTOR MED, 11 September 2013 - 12:30 PM.

[jerryshelly1]

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).

 

Good Luck.

Edited by jerryshelly1, 12 September 2013 - 09:26 AM.