Preparing a master mix
For HRMA with the LightScanner® master mixes and individual components are available for purchase. However we do not use master mixes. There are two major reasons. One reason is to reduce a price of runs, the second – to make more flexible assays. If you want to make an assay with unlabeled probes you should change Phire® Hot Start DNA polymerase and a corresponding buffer with Phusion® High Fidelity DNA polymerase with a supplied buffer to prevent degradation of probes during PCR. Usually optimization includes only gradient PCR but in difficult cases you can add DMSO (up to 10%), vary Mg2+ concentration. Individual components give you maximal flexibility.
File for the master mixes and components is here.
| With master mix | Without master mix | |
|---|---|---|
| components | all except primers | all separately: LCGreen Plus+, Phire polymerase, 5x buffer for the polymerase, dNTPs (Fermentas), primers |
| storage | -20°C; after the first use +4°C | -20°C exceptLCGreen Plus+ -20°C storage; after the first use +4°C |
| Polymerase | ? | Phire® Hot Start DNA polymerase |
| 5′-exonuclease activity | No | weak (do not use for assays with LUNA probes) |
| PCR conditions | 95°C 5′ 95°C 30” Ta 30” 72°C 30” 40-45 cycles 72°C 10′ 95°C 30” followed by 25°C 30” |
98°C 30” 98°C 5” Ta 5” 72°C 10” 40-45 cycles 72°C 1′ 95°C 30” followed by 25°C 30” |