Prbbbb:dna measurement on plate v1: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Line 34: | Line 34: | ||
<math> | <math> | ||
c = frac{(A-A0) \times e}{d} | c = \frac{(A-A0) \times e}{d} | ||
</math> | </math> | ||
Revision as of 14:05, 28 May 2009
Overview
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% deviation.
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.
Materials
- 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
Calculation
parameters
- well dimension: flat bottom 3.3 mm x 3.3 mm
- DNA extinction coefficient e: 50 [(ng * cm) / µl]
- path length in mm (d') or in cm (d):
d' = V / (3.3^2 * mm^2) d = d'/10
(V... volume per well in µl)
formula
[math]\displaystyle{ c = \frac{(A-A0) \times e}{d} }[/math]
- 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
Procedure
- 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
Notes
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.
References
Contact
or instead, discuss this protocol.