UBL Checklist Protocol

for

DNA Quantification ñ By Spectrophotometry

Revised May 2002

1.Ý Turn on the spectrophotometer (Jasco V530 or V550) and the computer that runs it.Ý Allow the spec to warm up for 15 minutes before use.

...

2.Ý In a microfuge tube, make a 1:10 dilution of your DNA sample by adding 10uL of DNA to 90uL of autoclaved Nanopure water.

3.Ý Take out two quartz cuvettes and wash them out with DI water:

ÝÝÝÝÝÝÝÝÝÝÝ ý Fill cuvette about æ full with DI water, pipet up and down several times with a P100 pipetor

ÝÝÝÝÝÝÝÝÝÝÝ ý Remove the water using the suction device attached to a pipet tip (change the pipet tip between samples!)

4.Ý After the spec has warmed up, start the scanning program by double-clicking on the 'Spectra Manager' icon on the desktop.

5.Ý Double-click on the 'Fixed Wavelength Measurement' option.Ý The scan window will come up.

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6.Ý Refill both cuvettes with DI water.Ý Place one cuvette in the 'R' (Reference) slot of the spectrophotometer, place the other in the 'S' (Sample) slot.Ý Close the sample cover.

ÝÝÝÝÝÝÝÝÝÝÝ ý Make sure there are no bubbles in the cuvettes ñ bubbles will distort the scan!

ÝÝÝÝÝÝÝÝÝÝÝ ý To remove bubbles, tab the cuvette gently with your finger.

7.Ý Click the 'Auto Zero' button on the scan window.

8.Ý Remove the cuvette from the 'S' slot, remove the water by suction, then fill with your diluted DNA.

ý Again, make sure there are no bubbles in the cuvettes and change the suction tip between samples.

9.Ý Place the cuvette with the sample back into the 'S' slot and close the cover.

10.Ý Click the 'Start' button on the scan window.Ý After a few moments, the absorbance will be displayed.Ý

ýÝ Check that the number you get is reasonable.Ý If not, double-check that there are no bubbles and scan again.

11.Ý Repeat steps 8-10 with your remaining samples.Ý Do not 'Auto-Zero' or 'Blank' the spectrophotometer between samples!

12.Ý When finished, record your absorbance data (A260), then rinse both cuvettes as in step 3.

13.Ý Shut down the computer and turn off the spectrophotometer.Ý Put away the cuvettes.

14.Ý Calculate the concentration of your DNA (taking into account the 1:10 dilution that you made):

1 A260 O.D. Unit for dsDNA = 50 ug/mL................................Example: A 1:10 dilution of dsDNA gives an A260 of 0.063

1 A260 O.D. Unit for ssDNA = 33 or 50 ug/mL......................................The concentration of DNA would then be

1 A260 O.D. Unit for RNA = 40 ug/mL...................................................0.063 x 50ug/mL x 10

Alternatively, you can use Beer's Law: a = e c l, where a = absorbance (A260), e = extinction coefficient, c = concentration, and l = path length of the scan (1 cm for our specs). Make sure your units match!

e is 20 g-1cm-1L for dsDNA, 30 g-1cm-1L for ssDNA, and 25 g-1cm-1L for RNA.