Quantitative real-time PCR (qPCR) is fast replacing conventional reverse transcriptase PCR in many molecular biology labs, increasingly reflected by high quality journals adjusting their requirements to only qPCR for gene expression data. qPCR relies on fluorescent DNA binding dyes which can be detected at specific excitation and emission spectra during the course of a PCR cycle. The ability to monitor the level of fluorescence on a cycle by cycle basis has led to this technique being labelled as both quantitative and real-time. This is in contrast to standard reverse transcriptase PCR which can be classified as end-point PCR, i.e only the final level of transcript can be measured semi-quantitatively by agarose gel electrophoresis.
With an increase in demand for quantitative, real-time gene expression data comes the need for sophisticated and user-friendly kits which can enable highly reproducible quantitative gene expression analyses. It is precisely in this niche market that Sigma’s SYBR® Green JumpStart™ Taq ReadyMix™ has made its mark. Ready mixes are commonplace in the qPCR market. They are popular because they eliminate the potential variability and increased work load that comes with pipetting to prepare in-house master mixes for each PCR experiment. Sigma’s SYBR Green ReadyMix achieves this as well as any other comparable kit that we’ve researched and even tried. Pleasingly, it achieves this despite the lower costs associated with it.
Sigma’s SYBR-Green JumpStart ReadyMix requires the addition of just the primers and template cDNA and contains antibody inactivated Taq which enables a hot start option and thus, does not allow non-specific binding to occur during the set up stages. The ReadyMix has therefore been optimized with the addition of the buffer and salts to enable highly efficient qPCR. The kit comes with extra MgCl2 in case the user wants to adjust the stringency of their reactions. Care has to be taken to use very clean preparations of cDNA and primers in the qPCR reactions. We usually dissolve both in sterile, distilled (ultraclean) water and avoid repeated freeze-thaw cycles. In addition, we do all qPCR reaction set ups either under a laminar flow hood or using the DNA handling robot from Qiagen.
Sigma provides their ReadyMix as a two times concentrate so it just requires the addition of sterile water to dilute it down to a working concentration. One inconvenient aspect of the Sigma ReadyMix is the size of the tube that it is provided in. Unlike other standard PCR reagents which are provided in 1.5ml or 2ml tubes that fit easily in standard freezer boxes, the ReadyMix tube is about double the size. It would’ve been more user-friendly to provide it in smaller aliquots. We store the tube separately in the freezer, which has made the organization of our PCR reagents more chaotic, and this is particularly a problem when multiple lab users are accessing the same reagents. We considered making our own aliquots but decided against it since we wanted to keep the ReadyMix as clean as possible. The tube size is compatible with PCR robots however, since there is a larger slot designed in them for master mix tubes of larger volume. We have recently purchased the Qiagen PCR robot and are pleased that the tube size wasn’t an issue for it.
The hot start feature kicks in above 70°C and this is a very important feature for all qPCR mixes because non-specific amplification can be very detrimental to interpreting accurate CT values. We are primarily conducting gene expression profiling of Arabidopsis plants that have been treated with pathogen elicitors to study defense gene signaling. Some genes are induced very slightly so it is imperative that our qPCR experiments are highly sensitive and reproducible. All in all, our lab has been very happy with the SYBR-Green kit from Sigma because it offers all the benefits of most kits on the market, and does so at an affordable price.
SYBR® Green JumpStart™ Taq ReadyMix™ from Sigma-Aldrich
An inexpensive and robust master mix for qPCR with added hot start Taq benefits.
The tube size is a bit awkward to store in standard freezer boxes but the product delivers on all technical grounds.
The Bottom Line
Sigma delivers yet another robust, easy to use, reproducible product for high throughput genomics.