Prbbbb:dna measurement on plate v1: Difference between revisions
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| Line 19: | Line 19: | ||
* DNA extinction coefficient e: 50 [(ng * cm) / µl] | * DNA extinction coefficient e: 50 [(ng * cm) / µl] | ||
* path length: | * path length in mm / in cm: | ||
d = V / (3.3^2 * mm^2) | d' = V / (3.3^2 * mm^2) | ||
V... volume per well in µl | d = d'/10 | ||
**V... volume per well in µl | |||
'''formula''' | '''formula''' | ||
| Line 27: | Line 28: | ||
c = (A * e) / d | c = (A * e) / d | ||
* c... DNA concentration in ng / µl | ** c... DNA concentration in ng / µl | ||
* A... Absorbance @ 260 nm | ** A... Absorbance @ 260 nm | ||
* d... path length in cm | ** d... path length in cm | ||
==Procedure== | ==Procedure== | ||
Revision as of 07:23, 20 May 2009
Overview
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 / in cm:
d' = V / (3.3^2 * mm^2) d = d'/10
- V... volume per well in µl
formula
c = (A * e) / d
- c... DNA concentration in ng / µl
- A... Absorbance @ 260 nm
- 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 (compare to reference)
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.
