HRMA with the LightScanner® requires specific reagents, enzymes and plastic.

The fluorescent dye LCGreen Plus+ is aliquoted into several vials each containing dye sufficient for 200 10 ul reactions. After the first use LCGreen Plus+ has to be stored at +4°C.

It is recommended to use Phire® Hot Start II DNA polymerase because LCGreen Plus + can inhibit the polymerase activity of regular Taq enzyme. Also Phire® insures stable polymerization even when different contaminants are present in the PCR. The polymerase enables quick cycling what can speed up genotyping. However, Phire® polymerase has weak 5′-exonuclease activity therefore it cannot be used for assays with Luna® probes. In case of the unlabeled probes you should use Phusion polymerase or master mix which already contains Phusion. Polymerases and buffers are stored at -20°C.

HRMA is a fluorescent based assay and it relies on a very precise measurement of fluorescence intensity. Therefore plates used for the analysis should have wells of white plastic with low level of background fluorescence. We use 96-well black microtiter plates with white wells. Plates can be covered with a foil seal to prevent degradation of LCGreen Plus+.

It is very important to overlay PCR mix with mineral oil. This is done in order to reduce technical variation while performing PCR and, more important, to prevent evaporation of your sample during scanning (temperature raises up to 98°C). It is recommended to use about 15-25 ul (equal volume) of mineral oil (Sigma M5904) to put on the top of 10 ul reaction.

Location:

Enzymes and buffers can be found in -20°C fridge in W325 in the box labeled as “LightScanner”

LCGreen Plus+ is in +4°C fridge in W326 in the box labeled as “LightScanner”

Plates and foil seals are in W330 on the shelves behind the LightScanner®

Mineral oil is on the shelf behind the LightScanner® in aliquots of 10 ml.

When you take a new vial of the enzyme, LCGreen+, the oil, plates or seals please fill in the withdrawal list next to the LightScanner®.