The following primers from primer plate LTP01 will be used for analysis of Rick’s Lake Trout DNA plate (from 4/28/2009): A1, C1, H1, B2. So, that’s 4 primer sets x 96 DNA samples = 384. HRM set up is here. A 1:10 dilution plate of Rick’s Lake Trout DNA1 plate (from 4/28/2009) was made for HRM. This means approximately 20ng of DNA used in each rxn. The robot was used to add 1uL of DNA from the 96-well plate to the appropriate wells of the 384-well HRM rxn plate. Plate was spun to collect the DNA at the bottom of the wells. The wells were visually inspected to ensure that each received the DNA. 1uL of DNA was manually added to those wells that did not receive sample (B23, C23).
The master mix for each primer set was then manually dispensed to the appropriate wells in the 384-well HRM rxn plate using a “matrix” auto-pipette. The real-time PCR was run in a Roche LightCycler480 machine with the following cycling paramters:
95C - 15mins
45 cycles of:
95C - 10s
60C - 15s
72C - 25s
After the completion of the real-time run, the plate was put through the High Melt Curve protocol.