Related Product Information
The Discoverase™ dHPLC DNA Polymerase enzyme mixture and buffer formulation have been optimized for use with denaturing high-performance liquid chromatography (dHPLC) systems(4). They were developed and tested using the Transgenomic WAVE® System.Discoverase™ dHPLC DNA Polymerase is supplied at 1 unit per µl.
| ||Kit Size|
|Component||100 rxn||500 rxn|
|Discoverase™ dHPLC DNA Polymerase||100 µl||500 µl|
|5X Discoverase™ PCR Buffer||1 ml||5 × 1 ml|
|50 mM Magnesium Sulfate (MgSO4)||1 ml||1 ml|
1. Add the following components to a sterile microcentrifuge tube on ice:*
|5X Discoverase™ PCR Buffer||10 µl||1X|
|10 mM dNTP mixture||1 µl||0.2 mM each|
|Primer mix (10 µM each)||1 µl||200 nM each|
|Template DNA x µl||(as required)|
|Discoverase™ DNA Polymerase ||1 µl||1.0 unit|
|Sterile, distilled water||to 50 µl||—|
*For multiple reactions, prepare a master mix of components common to all reactions to minimize reagent loss and enable accurate pipetting.
2. Mix contents of the tubes.
3. Cap the tubes and centrifuge briefly to collect the contents.
4. The following cycling protocol is recommended as a starting point, and may need to be optimized for different thermal cyclers. Note that the optimal annealing temperature is typically 5°C below the Tm of the primers.
1 min per kb
5. Maintain the reaction at 4°C after cycling. The samples can be stored at -20°C until use.
6. Proceed to dHPLC analysis
- Keep all components, reaction mixes, and samples on ice. After preparation of the samples, transfer them immediately to a preheated thermal cycler (94°C) and start the amplification program.
- Thin-walled reaction tubes are recommended
- One microliter of enzyme (1 unit) is appropriate for most targets.
- The annealing temperature of the reaction will vary depending on the Tm of your primers. The optimal annealing temperature is typically 5°C below the Tm of the primers.
2. Barnes, W.M. (1994) Proc. Natl. Acad. Sci. USA 91, 2216.
3. Tindall, K.R. and Kunkel, T.A. (1988) Biochemistry 27, 6008.
4. Xiao, W. and Oefner, P.J. (2001) Denaturing high-performance liquid chromatography: A review. Hum Mutat. 17, 439–74.