Picogreen Protocol to quantify DNA on the Analyst AD

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• Take picogreen out of fridge to start melting.

Can put in heat block at low temp (<30C) if necessary

• Turn on analyzer AD in Core Lab (WRRB 207) to let it warm up 30 min. prior to use.

Make sure analyzer has correct filters installed (it should)

Excitation: 485-20nm

Emission: 530-25nm

Dichroic: 505

• Make picogreen solution

mix up within a few hours of use.

Calculate the amount you will need. For full plate (96), mix up: 100 ul pico green

19.9 ml 1x TE pH 7.5

ie a 200 fold dilution in 1X TE pH 7.5 (1ul picogreen in 200 ul final volume)

• add 200 ul picogreen/TE mix to each well of black plate
• add 1-2 ul standard set to first and last row
• add same amt sample to other wells
• mix by pipetting (try not to introduce air bubbles)
• incubate DNA in picogreen/TE mix for at least 5 minutes in dark

• Go to Analyst AD

• Double click on CriterionHost
• save data as text or excel file on floppy
• analyze data in excel

use your standards to plot dna concentration against fluorescence

Use equation of a line y=mx+b to get ng/ul. Then solve for x and apply equation to all fluorescence readings and you will get ng/ul for all samples

ORDERING INFO for Plates:

96 well Corning plates. Always use black plates: 96 (P/N 42-000-0117) and 384 (P/N 42-000-0118) low volume plates from molecular devices or corning plates

Corning 384 well black Flat bottom Polystyrene not treated Microplate, 25 per Bag, without Lid, Non-Sterile (#3710)

Corning 96 Well Black Flat Bottom Polystyrene Not Treated Microplate, 25 per Bag, without Lid, Non Sterile (Item #3915)

-assay volume 75-200ul -well depth is 10.67

 

Make standards:

Note: TE used in procedure is pH 7.5

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