Integrated DNA Technologies
1055770
PrimeTime® Gene Expression Master Mix
Expression of genes involved in bile stress in E. coli were analysed by performing probe based multiplexed qPCR assays using probes and primers for each target synthesized from IDT and as well using ready use 2X Prime Time Gene Expression master mix from IDT. Assay performed well with ready to use master mix and at the end relative quantification across three biological replicates were consistent. This ready to use master mix contains all the components ready to go with assay except you add your primers and probes at required concentrations.
Relative quantification of gene expression by qPCR
cDNA synthesized from total RNA of E. coli exposed to bile stress
I have performed multiplexed qPCR assay using probe-based chemistry. Probe and Primer pair for each target were synthesized from IDT and then used ready to use 2X prime time gene expression master mix for reacitons (10 microliter volume). I have followed standard cycling conditions and primer and probe concentrations mentioned in the kit's protocol. I used probes at 200 nM and primers at 500 nM final concentrations in the 10 microliter reaction. Assay went well and Ct values were reproducible among three biological replicates.
For preparation of primers and probes stock solution, use IDT recommended buffer. This will help for long term storage without probe/primer degradation.
Relative expression of target genes were quantified. Satisfactory results were found. Prime time gene expression master mix has ideal concentrations of hot-start DNA polymerase; dNTPs; MgCl2; enhancers; and stabilizers. This saves lot time while setting up a plate and also reduces chances of pipetting error.
None
Easy to perform and simple protocol. Not expensive when compared to other sources.
I prefer to use this master mix for my probe based multiplexed qPCR assays.