Bio-Rad offers the iQ SYBR Green Supermix which contains dNTPs, 50 U/ml iTaq DNA polymerase, 6 mM MgCl2
, SYBR Green I, 20 nM fluorescein. To this mixture, just add your primers and template DNA to start the reaction. That all of the essential reagents are premixed in this Supermix removes the errors that can result when pipeting various solutions in small quantities. Since minimal pipeting is involved, the preparation time is considerably reduced and results are consistent.
The recommended reaction size for this mix is 50ul total volume. However, we have had consistently good results using just 25ul volumes. This is of considerable advantage since we could use the reagents for twice as many reactions as suggested by Qiagen. We have used iQ SYBR Green in our laboratory for almost 10 years along with the MyiQ icycler from Bio-Rad and we have had excellent results with this reagent. We have used this reagent to quantify genes of interest from primary samples such as mouse and human fetal and adult tissues as well as secondary cell lines. We have used this kit to quantify viral genes which were relatively low copy number. Although we have not quantified the absolute copy number of the viral genes, we have seen that the viral genes were less than 0.001% of cellular control genes such as beta-actin.
Although this kit works well for most genes, the fact that all the ingredients are pre-mixed becomes a disadvantage under some circumstances. For example, we had to amplify certain viral genes present in some diseased tissue. We were able to achieve our results by manipulating the concentrations of Mg2+ and the buffers. Such alterations are not feasible with a premixed solution. On occasion, there is amplification of genes that are not of interest which causes erroneous results due to noise. This noise is not due to the Supermix itself, but removal of noise in these reactions requires manipulation of the ingredients which again is not feasible with a premixed solution.
However, in summary, the iQ SYBR Green Supermix from Bio-Rad is an excellent kit for quantification of most genes using real-time PCR. This kit works well when using iCycler real-time PCR machine from Bio-Rad.
iQ SYBR Green Real-Time PCR Supermix from BioRad
All the ingredients needed for real-time PCR except the primers and the cDNA are premixed. This reduces the error rate since individual components are not pipeted separately.
Manipulation of constituent reagents is not feasible.
The Bottom Line
SYB R Green Supermix works well for quantification of most genes.