Protocol
EpiMelt Real-Time PCR Master Mix
EpiMelt Real-Time PCR Master Mix
High specificity ready-to-use mix for real-time Hot Start PCR with EvaGreen®.
Optimized for epigenetic analysis using HRM technology. 2x concentrated.
For 200 & 500 reactions in 20 μl
| Catalog # | Size |
| # EPI – qPCR – 200 | 200 reactions in 20 μl |
| # EPI – qPCR – 500 | 500 reactions in 20 μl |
Store at -20°C upon arrival
User manual version 2. Last revision date: 03-08-2023 EvaGreen® is a registered trademark of Biotium Inc.
For research use only. Not for use in diagnostic procedures.
Download the Protocol here:
EpiMelt Real-Time Master Mix
| 200 Reactions | 500 Reactions | Storage | |
| EpiMelt Master Mix 2x | 2 x 1 mL | 5 x 1 mL | -20°C, in darkness |
| EpiMelt Sterile Water | 2 x 1.5 mL | 5 x 1.5 mL | -20°C |
Table 1. Contents of the EpiMelt Real-Time PCR Master Mix.
EpiMelt Master Mix 2x composition
| Component | Amount |
| EpiMelt Hot Start DNA Polymerase | 0.1 U/μl |
| MgCl2 | 4 mM |
| dNTPs | 0.5 mM of each dNTP |
| 2x reaction buffer with EvaGreen® |
Table 2. EpiMelt Master Mix x composition.
Additional equipment and reagents
- EpiMelt Bisulfite Modification Standard Kit (MethylDetect)
- Vortex
- Microcentrifuge
- Real-time thermocycler
Important notes
- Before use, all solutions should be thawed thoroughly on ice, gently mixed by inverting, and briefly
centrifuged - Up to 3x repeated freeze-thaw cycles do not influence the activity of this product
For research use only. Not for use in diagnostic procedures.
Protocol
The EpiMelt Bisulfite Modification Standard Kit (MethylDetect) is recommended for the bisulfite conversion
of template DNA.
- Thaw EpiMelt Master Mix 2x and Sterile Water on ice, gently mix by inverting, and briefly centrifuge. Keep on ice.
- Prepare the PCR mix by adding the following components in the order listed below:
| Component | PCR reaction volume | PCR reaction volume |
| 10 μl | 20 μl | |
| EpiMelt Master Mix 2x | 5 μl | 10 μl |
| Primer 1** | 0.1 – 1 μM* | 0.1 – 1 μM* |
| Primer 2** | 0.1 – 1 μM* | 0.1 – 1 μM* |
| DNA template | 3 ng – 1 μg | 3 ng – 1 μg |
| Sterile Water | Up to 10 μl | Up to 20 μl |
Table 3. Components of the PCR mix.
*: Recommended for standard real-time PCR.
**: Final concentration in the reaction mixture
- Gently vortex the samples and briefly centrifuge to ensure that the content is collected at the
bottom of the tube. - Place the tubes in the thermocycler and start the PCR program.
Example of an amplification profile:
| Step | Temperature | Time |
| Initial denaturation | 95°C | 10 min |
| 35 – 50 cycles | 95°C 50 – 68 °C 72 °C |
15 – 30 s 30 – 60 s 15 – 60 s* |
Table 4. Example of an amplification profile.
*: Depending on the length of the PCR product, for products >500 bp 1 min PCR product melting analysis is recommended
Download the Protocol here:
Recommended ROX mixture
HiROX (0.6-1 μL per 50 μL of total reaction volume): Applied Biosystems: 7000, 7300, 7700, 7900HT
Fast, StepOne, StepOnePlus.
LoROX (0.6-1 μL per 50 μL of total reaction volume): Applied Biosystems: 7500, Stratagene: Mx3000P,
Mx3005P, Mx4000P
Ordering Information
MethylDetect offers a number of EpiMelt Methylation Detection Assays targeting specific genomic regions. For a complete overview of the products and ordering information, please visit www.MethylDetect.com.
Supplementary Information
License disclaimer
For patent license limitations for individual products, please refer to www.MethylDetect.com.
Regulatory disclaimer
For Life Science research only. Not for use in diagnostic procedures.
EvaGreen® is a registered trademark of Biotium Inc.
Safety data sheet
Please follow the instructions in the safety data sheet (SDS) at www.MethylDetect.com.
Contact and support
Please refer to www.MethylDetect.com.
For research use only. Not for use in diagnostic procedures.
©2019 MethylDetect. All rights reserved.