PicoGreen dsDNA Quantitation

From Genome@Yale

for 10 samples (must make more WS, if more than 10 samples are measured)

need special Vial Inserts ("cuvettes"), Volume: 0.25mL Wheaton Science Products No.:225350-531 (Fisher Cat # 03-337-51)

1. Make PicoGreen working solution (WS) by diluting 5uL of PicoGreen into 995uL TE (200 fold dilution) - enough for 7 standards and 3 samples
2. Make standards: make 100uL 2ng/ul and 100uL 20pg/uL genomic DNA solution (Input DNA from ChIP)
3. Prepare serially diluted standards according to the table below
4. Add 2uL of sample and 98uL TE and 100uL WS (100 fold dilution)
5. Place Blue module into the Turner Biosystem Fluorometer/Luminometer and turn on the machine
6. Launch the SIS Excel module on the desktop
7. Obtain round cuvettes and transfer 100uL of DNA-PicoGreen mix to a cuvette and measure the fluorescence
8. Repeat the previous step until all samples are read
9. Substract the background fluorescence from #0 from all readings
10. Generate a standard curve using trend fit in Excel
11. Determine the concentration of ChIP DNA (remember to multiply by the dilution factor, ie 100)

Standards
tube  conc pg/uL    stock     TE     PicoGreen WS
0     0             0         100    100
1     2.5           25  lo    75     100
2     5             50  lo    50     100
3     10            1   hi    99     100
4     25            2.5 hi    97.5   100
5     50            5   hi    95     100
6     100           10  hi    90     100