SensiFAST™ HRM Kit

Cat No. Size List Price* Qty  
BIO-32002 200 Reactions
$188.00
 
BIO-32005 500 Reactions
$454.00
 
BIO-32020 2000 Reactions
$1,549.00
 
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*For more information on pricing please contact us

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SensiFAST HRM uses the latest advances in buffer chemistry and enhancers, together with an antibody-mediated hot-start DNA polymerase system, to ensure that the SensiFAST HRM Kit delivers fast, reproducible, highly-specific and ultra-sensitive HRM analysis.

Features & Benefits

  • Ultra-sensitive: accurately discriminates class 4 (A/T) SNP mutations
  • Rapid - enabling accurate results to be achieved faster
  • Highly reproducible - highly-specific, high-resolution melt analysis
  • Optimized - assays quickly and reliably established, even for difficult genomic loci

Instrument Compatibility

ABI 7300, 7500 FAST, Qiagen Rotor-Gene™ 6000, Eppendorf Mastercycler® ep realplex, Illumina® Eco™, Roche LightCycler®; 96 and 480, Bio-Rad Opticon®, CFX96™, CFX384™, Idaho LightScanner® 32 (LS32™), Takara Thermal Cycler Dice (TP800) (See product selection table).


Description

High Resolution Melt (HRM) curve analysis is a post-PCR analysis method of characterizing nucleic acid samples based on DNA strand dissociation behaviour during transition from double-stranded DNA to single-stranded DNA with increasing temperature. It uses intercalating dyes, highly accurate melt curves and the application of specific statistical analyses.

SensiFAST HRM has been developed using the latest advances in buffer chemistry and enhancers, together with an antibody-mediated hot-start DNA polymerase system, to ensure that the SensiFAST HRM Kit delivers fast, reproducible, highly-specific and ultra-sensitive HRM analysis, ideal for small and medium-sized genotyping projects.

The combination of our SensiFAST DNA Polymerase, buffer and EvaGreen® dye enables amplification and discrimination of even the most challenging sequence differences, such as class 4 SNPs (Fig. 1) without sequence preference. Main applications of HRM include Single Nucleotide Polymorphisms (SNPs) genotyping, epigenetics (DNA methylation analysis), zygosity testing (DNA mapping and DNA fingerprinting) and gene scanning (search for the presence of unknown variations in PCR amplicons).


Notes

SensiFAST is a trademark of Bioline Reagents Ltd.


Components

Reagent

200 x 20µl reactions

500 x 20µl reactions

2000 x 20µl reactions

SensiFAST HRM  mix (2x)

2 x 1ml

5 x 1ml

20 x 1ml


Volume

IO-32002: 200 x 20µl Reactions: 2 x 1ml
BIO-32005: 500 x 20µl Reactions: 5 x 1ml
BIO-32020: 2000 x 20µl Reactions: 20 x 1ml

Storage & Stability

All kit components should be stored at -20°C upon receipt. When stored under the recommended conditions and handled correctly, full activity of the kit is retained until the expiry date on the outer box label. Avoid exposure of the EvaGreen to light.


Shipping conditions

SensiFAST HRM Kit is shipped on dry/blue ice.


Certificates of Analysis (COAs)

Certificates of Analysis for SensiFAST™ HRM Kit are grouped by catalogue number and then listed by batch number. Larger pack sizes are listed first. To locate the COA for your product, first find the catalogue number and then the required batch number.

View all 19 COAs for SensiFAST™ HRM Kit →


Frequently asked questions

  • What is the advantage of working with SYBR Green I?

      SYBR Green I is an inexpensive, universal dye which binds to all dsDNA. It can be easily used in combination with a simple primer pair to detect PCR products in real-time. This dye is very attractive for researchers analysing lots of different genes.

  • What is the advantage of working with a probe system?

      The probe system is always specific; only detect the gene of interest. It is also possible to distinguish between similar sequences with small differences like SNPs or mutations. In general, probe assays need less optimisation than SYBR Green I assays.

  • Can I use a SensiFAST Kit for standard real-time PCR?

      Although SensiFAST kits have been designed for fast PCR on the new generation of fast machines, they will work equally well for standard or fast PCR protocols on all real-time PCR machines.
  • Can I use a SensiFAST SYBR Kit for a probe assay?

      This is not possible because the SYBR is pre-mixed into the SensiFAST SYBR mastermix.
  • Why do the SensiFAST kits contain a hot-start Taq polymerase?

      Polymerase activity during the reaction set-up causes non-specific amplification including primer-dimer formation. To avoid non-specific amplification the polymerase is only activated after heating at 95°C for 2-3 minutes.
  • Why do you no longer sell kits contain dUTP (and UNG)?

      This is because it is because it is only effective if all the researchers either doing PCR in the laboratory or using the same thermocycler are also using a dUTP/dNTP and UNG system. If even one researcher is not, it ceases to be an effective control. Using dUTP has also been shown to be inefficient as it increases the Ct values by reducing reaction efficiency. Most labs do not need to use the dUTP method of control, optimised protocols will allow high specificity of PCR and good lab practices (using disposable consumables, the use of filter tips and maintaining a separate area for PCR set-up and PCR amplification and any post-PCR analysis) virtually eliminate the risk of cross contamination.
  • Why do certain kits contain a ROX passive reference?

      The emission recorded from ROX during the baseline cycles is used to normalize the emission recorded from the reporter (SYBR) in later cycles in some instruments. ROX compensates for small fluorescent fluctuations such as bubbles and well-to-well variations that may occur. Each of these instruments having the capacity to analyze the real-time PCR data with the passive reference signal either on or off.
  • Why are the ROX concentrations different?

      The amount of the ROX passive reference dye needed varies depending on the instrument optics, our SensiFAST kits have been optimised for these different instruments (see Product Selection Tool).
  • What is the difference between using ROX and using fluorescein?

      Fluorescein is an alternative passive reference dye used just in the Bio-Rad instruments (see Product Selection Tool), the SensiFAST SYBR & Fluorescein contains fluorescein premixed in the mastermix at optimised concentrations.
  • Is the SYBR in a separate tube?

      For your convenience the SensiFAST kits have an optimised amount of SYBR Green in the mastermix.
  • What is the difference between a one-step and a two-step real-time PCR reaction?

      In a one-step reaction the reverse transcription reaction and the real-time PCR reaction are done in one tube, making this a closed tube assay, so contamination can be avoided. It saves pipeting steps and time, and is easy in handling, making it ideal for high throughput screening.

      In a two-step reaction the reverse transcription reaction and the real-time PCR reaction are done in separate tubes. It gives a more flexible way of working in that the cDNA can be used for more than one real-time PCR reaction and can be archived, eliminating the need to continually isolate RNA. For convenience Bioline sells the SensiFAST cDNA Synthesis Kit separately if you wish to take a two-step approach.
  • What template (RNA/cDNA) is this compatible with?

      SensiFAST One-Step Kits can be used with most RNA/cDNA templates. To give you some idea of a few of the types of templates used with these kits:

      Human - Rosato R.R., et al. Cancer Res. 67: 9490-500 (2007)
      Rat - Remund K., et al. Expt. Lung Res. 35: 359-370 (2009)
      Drosophila - Brown A.E., et al. PloS ONE 4: e4490 (2009)
      Nitrogen-fixing bacteria - Bahlawane C., et al. Mol. Plant-Microbe Inter. 21: 1498-1509 (2008)
      Diphtheria bacteria - Jochmann N., et al. Microbiology 155: 1459-1477 (2009)
      Green algae - Wobbe L., et al. PNAS 106: 13290-13295 (2009)
      Bovine virus - Park S-I., et al. J. Virol. Methods. 159: 64-6 (2009)

      For cDNA/DNA templates SensiFAST SYBR and Probe kits are used. To give you some idea of a few of the types of templates used with these kits:

      Stem Cell - Bernardo A.S., et al. Stem Cells 27(2): 341 -351 (2008)
      Cow - Baumert A., et al. J. Dairy Research 76(3): 356-364 (2009)
      Mouse - Hoyles R.K., et al. Arthritis Rheum. 58(4): 1175-88 (2008)
      Quail - De Winter P., et al. British Poultry Science 49(5): 566 – 573 (2008)
      Insect - Bass C., et al. Malaria journal 6 111 (2007)
      Fish - Miller M.R., et al. J. Nutr. 138(11): 2179-2185 (2008)
      Crab - Wilcockson D.C. & Webster S.G. Gen. & Comp. Endo. 156: 113–125 (2008)
      Nematode - Murray S.L., et al. Mol. Plant-Microbe Interactions 20(11): 1431–1438 (2007)
      Plant - Hecht V., et al. Plant Physiology Preview DOI:10.1104/pp.107.096818 (2007)
      Yeast - Kawauchi J., et al. Genes & Dev. 22: 1082-1092 (2008)
      E.coli Bacteria - Saeed H.A., et al. Research J. Microbiology 4(4); 173-177 (2009)
      Virus - Muscillo M., et al. Water, Air, & Soil Pollution 191: 1-4 (2008)



Other researchers use:


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