This mix was developed to quantify gene expression via real-time PCR using genomic DNA or cDNA as template. This mix includes:
Thermo-Start DNA Polymerase: A hot-start version of Thermoprime Plus DNA Polymerase, which prevents non-specific amplification during set up of the reaction. Note, that this enzyme requires 15 min of activation at 95°C.
Reaction buffer: Suitable for real-time and end-point analysis. This buffer includes 3M MgCl
2 in final 1X concentration. Also containes enhancers to enable amplification of a broad range templates, including plant DNA and DNA with high CG content. This buffer also includes inert blue dye for easier handling of the kit.
dNTPs: Include dTTP in order to increase sensitivity of the reaction.
ROX dye: Passive reference dye; supplied at final concentration of 500 nM.
SYBR Green: For detection of pcr product.
According the manual, this kit is compatible with cyclers that require 500 nM ROX dye concentration like ABI PRISM 7000, 7300, 7700, 7900 and 7900HT, including fast mode.
All the reagents should be thawed on ice and spun down to prior to use. The SYBR Green I mix should NOT to be vortexed.
In the manual, there is an example for setting up a 25 µl reaction. But minimum reaction volume should be chosen according the instrument requirements. I did the reaction on ABI 7900HT instrument and prepared mix according the kit's manual. Per one reaction the following components should be mixed: 12.5 µl of Absolute Blue SYBR Green mix, forward primer, reverse primer, cDNA template and water up to the final volume. Note that the optimal working concentration for each set of primers needs to be empirically determined. The manual suggests starting with a low primer concentration of 70 nM and then increasing it, as needed.
I tried this Absolute Blue SYBR Green mix with several genes using cDNA as template. I obtained satisfying results with the housekeeping gene GAPDH where the concentration of both primers was 100 nM. I spent a long time trying to find optimal concentration and reaction conditions, changing cDNA dilution and primer concentration from 70 nM up to 300 nM according to the manual for the other genes that I am working with, but finally the cost of my efforts and time that this setup was taking lead me to shift to use another SYBR Green mix for QPCR. I succeed very well with Invitrogen’s SYBR® GreenER™ Two-Step qRT-PCR Kit (I reviewed it previously). With Invitrogen’s mix, I am getting a good standard curve and the melting curve shows very low or no primer-dimer. The repeats of the same transcription reaction show statistically close results.
Neuroimmunology Laboratory Manager
Department of Neurology
Sourasky Tel-Aviv Medical Research Center