Download Plate reader quantitation with PicoGreen

Plate reader quantification of PCR amplicons with PicoGreen
1) Prepare 8 DNA standards using serial 2:1 dilutions from 10 ng/μl DNA Standard in
water in a strip of PCR tubes.
Concentration of standards should be as follows (in ng/ml):
10
5
2.5
1.25
0.625
0.312
0.156
0.078
2) Each plate can quantify a maximum of 88 samples. Use multiple plates, if required.
3) Prepare a master mix containing:
199 μl 1X TE for X samples + 8 standards/plate
1 μl PicoGreen fluorescent dye for X samples + 8 standards/plate
4) Vortex and aliquot 195 μl of master mix into X sample wells + 8 standards wells of a
black Fluotrac 200 plate (Greiner).
5) Add 5 μl of sample into each sample well, and 5 μl of the standards into each
standard well.
6) Attach a clear, optical film seal to the plate and press the seal down firmly with
rubber spatula.
7) Vortex the plate for 5 sec. Spin the plate at 930 rcf for 1 min. If any droplets remain
on the seal, spin again.
8) Put the plate in the plate reader, use the Multimode Analysis software (Beckman
Coulter) to enter the layout of samples/standards in the plate, enter the
concentrations of each standard, and run the machine.
9) Check the standard curve as a linear fit, and if satisfied save the resulting Excel
output containing sample concentrations to a flashdrive.