Difference between revisions of "Prbbbb:dna measurement on plate v1"
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Revision as of 08:01, 20 May 2009
Rather than "nanodropping" every well by hand, we measure DNA concentrations on a plate reader.
Accuracy The results agree with nanodrop measurements to within 5% accuracy.
Limits Concentrations down to 10 ng/µl can be reliably measured. The volume should be 20 µl or more per well. The samples are later recovered for dilution and storage.
- Tecan plate reader
- (or any reader that accepts 384 plates and can measure at 260 and 280 nm)
- 384 well UV transmissive plate with flat clear bottom
- we use Greiner UV star Cat # 781 801
- miniprepped DNA, at least 30 µl
- well dimension: flat bottom 3.3 mm x 3.3 mm
- DNA extinction coefficient e: 50 [(ng * cm) / µl]
- path length in mm / in cm:
d' = V / (3.3^2 * mm^2) d = d'/10
(V... volume per well in µl)
c = (A-A0 * e) / d
- c... DNA concentration in ng / µl
- A... Absorbance @ 260 nm
- A0...Absorbance of blank sample (same amount, same buffer/water)
- d... path length in cm
- Pipette exact volume of DNA samples into 384 well plate
- measure 260 nm and 280 nm absorbance on plate reader
- Pipette sample back into storage plate
- calculate DNA concentration from formula above
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
raik: This protocol is quick and robust in my hands.
or instead, discuss this protocol.