Storage and stability:
The SensiFAST SYBR No-ROX One Step Kit is shipped on dry ice/blue. All kit components should be stored at -20 ° C upon receipt. Excessive freezing/thawing is not recommended. When stored under optimal conditions, reagents are stable for a minimum of 6 months from the date of buys.
Bioline operates under the ISO 9001 Management System. The SensiFAST SYBR No-ROX One-Step Kit and its components are extensively tested for activity, processivity, efficiency, heat activation sensitivity, absence of nuclease contamination, and absence of nucleic acid contamination.
Harmful if swallowed. Irritating to eyes, respiratory tract, and skin. Consult the material on the safety data sheet for more information.
For research use only.
The SensiFASTTM SYBR No-ROX One-Step Kit has been formulated for the synthesis and subsequent real-time PCR in a single tube. A combination of the latest advances in buffer chemistry along with a transcriptase and hot-start DNA polymerase system ensures SensiFAST SYBR No-ROX One-Step Kit produces fast, highly specific and ultrasensitive single step RT-qPCR. The SensiFAST SYBR No-ROX One-Step Kit consists of a one-step mix of SensiFAST SYBR 2x, as well as a separate reverse transcriptase and RiboSafe RNase Inhibitor.
The sequence and concentration of the primers, as well as amplicon length, can be critical for specific amplification, performance, and overall efficiency of any RT-qPCR. We strongly recommend keeping in mind the following points when designing and running your RT-qPCR:
- Use primer design software, such as Primer3 or visual OMPTM. The primers must have a melting temperature (Tm) of about 60 ° C
- The optimal length of the amplicon should be 80-200 bp, and should not exceed 400 bp.
- The final primer concentration of 400nM is adequate for most SYBRGreen-based reactions, however, to determine the concentration we recommend titrating in the range 0.1-1M.
- Use an equimolar primer concentration.
- When possible, use intron-traversing primers to avoid amplification from genomic DNA.
- Template: It is important that the RNA template is intact and devoid of DNA or contaminating inhibitors of both transcription and PCR. For high purity RNA, we recommend using the Bioline ISOLATE RNA Mini Kit (BIO-52043). RNA reserves and dilutions should be made in water treated with DEPC (BIO-38030) to avoid any RNase-mediated degradation. The recommended amount of template for one-step RT-qPCR is
depending on the type of RNA used.
- Total RNA: purified total RNA can be used in the range of 1pg to 1g per 20l reaction.
- mRNA: purified mRNA from 0.01 pg per 20 µl can be used reaction.