Protocol Citation: Bioline 2016. SensiFAST™ HRM Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.fyrbpv6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: October 04, 2016
Last Modified: March 13, 2018
Protocol Integer ID: 3825
Abstract
The SensiFAST™ HRM Kit has been developed for fast, highly reproducible High Resolution Melt (HRM) analysis and has been validated on commonly used real-time instruments. A combination of the latest advances in buffer chemistry and enhancers, together with an antibody-mediated hot-start DNA polymerase system, ensures that the SensiFAST HRM Kit delivers fast, highly-specific and ultra-sensitive HRM analysis.
For ease-of-use and added convenience, SensiFAST HRM is provided as a 2x mastermix containing all the components necessary for real-time PCR, including the EvaGreen® dye, dNTPs, stabilisers and enhancers. As a ready-to-use premix, only primers and template need to be added.
Guidelines
Kit components
| Reagent | 200 x 20 μL reactions | 500 x 20 μL reactions | 2000 x 20 μL reactions | |
| SensiFAST HRM mix (2x) | 2 x 1 mL | 5 x 1 mL | 20 x 1 mL |
General considerations
To help prevent any carry-over DNA contamination, we recommend that separate areas be maintained for PCR set-up, PCR amplification and any post-PCR gel analysis. It is essential that any amplified PCR product should not be opened in the PCR set-up area.
Primers: The sequence and concentration of the primers, as well as amplicon length, can be critical for specific amplification, yield and overall efficiency of any real-time PCR. We strongly recommend taking the following points into consideration when designing and running your real-time PCR:
use primer-design software, such as Primer3 (http:// frodo.wi.mit.edu/primer3/) or visual OMP™ (http:// dnasoftware.com/). Primers should have a melting temperature (Tm) of approximately 60°C.
optimal amplicon length should be 80-200 bp, and should not exceed 400 bp
final primer concentration of 400 nM is suitable for most reactions, however to determine the optimal concentration we recommend titrating in the range 0.1-1 μM
use an equimolar primer concentration
Template: it is important that the DNA template is suitable for use in PCR in terms of purity and concentration. In addition, the template needs to be devoid of any contaminating PCR inhibitors (e.g. EDTA). The following should be considered when using genomic DNA templates:
Genomic DNA: use up to 1 μg of complex (e.g. eukaryotic) genomic DNA in a single PCR. We recommend using the Bioline ISOLATE II Genomic DNA Mini Kit (BIO-52066) for high yield and purity from both prokaryotic and eukaryotic sources
MgCl2: The MgCl2 concentration in the 1x reaction mix is 3 mM, which is optimal for SensiFAST HRM in the majority of real-time PCR conditions. If necessary, we suggest titrating MgCl2 to a maximum of 5 mM.
PCR controls: It is important to detect the presence of contaminating DNA that may affect the reliability of the data. Always include a no template control (NTC), replacing the template with PCR-grade water.
Troubleshooting Guide
See the Bioline full documentation for detailed troubleshooting instructions.
http://www.bioline.com/us/downloads/dl/file/id/2705/sensifast_hrm_kit_manual.pdf
Materials
MATERIALS
SensiFAST™ HRM KitBiolineCatalog #BIO-32002
Reaction mix composition
Reaction mix composition
Prepare a PCR master mix. The volumes given below are based on a standard 20 μL final reaction mix and can be scaled accordingly.
| Reagent | Volume | Final concentration | |
| 2x SensiFAST HRM Mix | 10 μL | 1x | |
| 10 μM Forward Primer | 0.8 μL | 400 nM | |
| 10 μM Reverse Primer | 0.8 μL | 400 nM | |
| H2O | up to 16 μL | ||
| Template | 4 μL | ||
| 20 μL Final Volume |
Suggested thermal cycling conditions
Suggested thermal cycling conditions
SensiFAST HRM Kit is compatible with either 3-step or 2-step cycling:
3 step cycle
| Cycles | Temperature | Time | Notes | |
| 1 | *95°C | *2 min | Polymerase activation | |
| 40 | 95°C 60-65°C 72°C | 5 s 10 s **5-20 s | Denaturation Annealing Extension (aquire at end of step) |
*2min for cDNA, 3min for genomic DNA
** Not recommended to extend beyond 20 seconds
2-step cycle
| Cycles | Temperature | Time | Notes | |
| 1 | *95°C | *2 min | Polymerase activation | |
| 40 | 95°C 60-65°C | 5 s **15-30 s | Denaturation Annealing/extension (aquire at end of step) |
*2min for cDNA, 3min for genomic DNA
** Not recommended to extend beyond 20 seconds
HRM analysis
HRM analysis
After the reaction has reached completion, refer to the instrument instructions for the option of melt-profile analysis.