Quantification of DNA using the Fluoroimager and Pico Green

1) Dilute template with ddH₂O to be within range of 0 – 500 ng/ul.

2) Make up standards. For a range of 0 – 500 ng/ul:

-use human DNA standard dilutions in frig. (see below)

-for each standard mix: 1ul pico green

5 ul standard

94 ul TLE (or TE)

-pipette into first (1) column of black plate (with transparent wells), highest concentration at top of plate, lowest concentration at bottom.

- more or less standard can be used depending on the concentration of your test samples. For example, if you use 1 ul of standard, you can test DNA of concentrations between 0 and 100 ng/ul. Always use 1 ul of pico green, but adjust the TLE accordingly.

How to make a dilution series. Mix very well before transferring between tubes. Procedure is assuming that you’re starting with Promega’s Human Genomic DNA (G-2304) standard that is at 190 ng/ul. If you are using a different standard, then adjust the volumes for Tube 1 accordingly.

	Tube contents	Concentration (ng/ul)
Tube 1	105.26 ul DNA 94.74 ul TLE	100
Tube 2	100ul of Tube 1 100ul of TLE	50
Tube 3	100ul of Tube 2 100ul of TLE	25
Tube 4	100ul of Tube 3 100ul of TLE	12.5
Tube 5	100ul of Tube 4 100ul of TLE	6.25
Tube 6	100ul of Tube 5 100ul of TLE	3.125
Tube 7	100ul of Tube 6 100ul of TLE	1.5625
Tube 8	100ul of TLE	0

3) Make up mastermix for samples

-add 1 ul pico green X the number of samples (allow a little extra, too)

-add 98 ul TLE (or TE) X the number of samples

-pipette 99 ul of mastermix into each well

-add 1 ul diluted template to each well

3) Place parafilm over top of plate. Gently vortex to mix solutions in wells.

-remove parafilm when done

4) Scan on fluoroimager on high sensitivity. Start microsoft EXCEL program up.

5) Open ImagequantTL, click array analysis (Imagequant TL v 2003 is only on Lab18)

6) Click on any file to open the program; you will get an error asking to restore the default image (click okay)

7) Open the file with the quantification plate

8) Click on the parameters tab in the bottom left corner and correct the number of columns and rows for your samples

9) Place the crosshairs in the middle of the upper left well and drag to the middle of the lower right well (even if it’s empty)

10) Make sure the circles are inside each well – if not, unclick the auto-size box and change the circle radius manually

11) Click next (don’t save the grid type)

12) Click parameters in the lower left and select negative controls

13) Click on the well containing your negative control

14) Click the symbol next to set negative control

15) Click next; click parameters

16) Click on a well containing a standard

17) Change the normalized volume to be the concentration of that standard (use average volume)

18) Click normalize

19) Look at the concentrations for your standards in the measurements window – if the concentrations are not correct pick a different standard that seems more accurate

20) Click on the measurements window and click export – you can now paste the data into excel

Quantification of DNA using the nanodrop (Medrano Lab)

This works even if someone is running the RT machine

1. Open Nanodrop from desktop

2. Make sure it is set to DNA-50 – if not, go to User Preferences -> Nucleic Acids -> Other -> Settable constant: 50

3. Clean pedestal with water and kimwipe

4. Put 2 ul of water/elution buffer onto measurement pedestal. Lower arm but don’t press it down.

5. Click blank

6. Clean pedestal with kimwipe

7. Add 2 ul of sample. Hit measure button.

You will have to record the measurements – they’re not stored on the computer