Reference dye (ROX) - (Apr/27/2005 )

Each reaction mixture will include the following:

12.5 μl 2X Stratagene Brilliant SYBR Green QPCR Master Mix (includes buffer, DNA polymerase, dNTPs, SYBR Green)
0.3 μl Forward primer at 25 μmol l-1
0.3 μl Reverse primer at 25 μmol l-1
0.38 μl Reference dye (ROX) (1:500 dilution)
10.52 μl Nuclease-free water
1 μl cDNA template



Should I add Reference dye (ROX) into internal reactions, sample reactions and standard reactions? What for?


Thanks.

Dear friend,

Yes. You should. ROX is a reference dye which will help you calibration the initial fluoresence reading of every tube.

For most machine, before you run any real-time PCR, the camera will try detect the presence of particular dye and normalised the flouresence reading of every single tube. This is to make sure that your every tube start reaction at the same flouresence unit.

However, the initial reading for SYBR green is very low, camera can't detect it. So you need to add in ROX, which has the same wave length with SYBR Green.

Don't worry about the ROX, It would not interfere with your reaction. ROX will blich in 10-15 cycles.

Thanks.

Should i also use ROX when i use Taqman, Sybgreen or hybridization probes? Does it matter which detection systems i use?

Dear friend,

Internal calibration dye only nececerly when you use SYBR green. TaqMan probe will always have some signal although with the presence of quencher. Because no quencher is 100% efficein, thus your TaqMan probe will release small amount of signal.

Best regards
Yong