The LightCycler® Fast Start DNA Master SYBR® Green I is convenient, reliable, easy to use and gives excellent reproducibility. I highly recommend it.
About 6 months ago, our lab decided to move to real-time PCR system, also known as quantitative PCR (qPCR). Our research department has the Roche LightCycler® 2.0, and I began to set-up the system using SYBR® Green qPCR by purchasing and using the LightCycler® FastStart DNA Master SYBR® Green for 96 reactions of 20 μl.
The kit contains one tube of LightCycler® Fast Start Enzyme, three tubes of LightCycler® SYBR® Green Master Mix and one tube of MgCl2 at a concentration of 25 mM. Also each kit has two vials with PCR grade water. The LightCycler® FastStart Reaction Mix is a ready-to-use reaction mix designed for real-time RCR assays using SYBR® Green as a detection agent on a LightCycler® 2.0 System. The formulation stores very well beyond the shelf life indicated by the company.
The FastStart Taq polymerase allows for hot start PCR. Using hot start has been shown to improve specificity and sensitivity of PCR by avoiding the formation of non-specific amplification products. The FastStart Taq polymerase has been modified to start amplification after activation at 95ºC for 10 min before beginning the amplification program.
PCR reactions may be set up at room temperature, but for optimal performance, the capillaries should be inserted into the cold block, which can be purchased separately from Roche. Everything needed for to set up reactions is provided, even molecular grade water. The major advantage of the master mix is that the reaction volume can be scaled down to 10 ul; this doubles the number of reactions that can be performed with the kit.
However, I found the following slightly inconvenient:
1. Preparation of a small number of the reactions. If you do not use all of the reaction mix, then you have to use the prepared mix during following week (stored at 4ºC) or freeze and thaw it. Although I didn't see a loss in sensitivity when freeze-thawing it (I tried this once), I prefer to aliquot the DNA Master SYBR® Green I Reaction Mix prior setting up PCR reaction.
2. 10 ul of the enzyme must be mixed with one vial of DNA Master SYBR® Green I Reaction Mix for every use. I would prefer to use pre-aliquoted enzyme to avoid pipetting mistakes and possible contamination.
3. The kit can only be purchased in 96 or 480 reaction sizes. Offering a kit in a size between these sizes would give the user a better purchasing option.
In conclusion, the LightCycler® Fast Start DNA Master SYBR® Green I is convenient, reliable, easy to use and gives excellent reproducibility. I highly recommend it.
Neuroimmunology Laboratory Manager
Department of Neurology
Sourasky Tel-Aviv Medical Research Center