Prbbbb:dna measurement on plate v1

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(Calculation)
(Calculation)
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==Calculation==
==Calculation==
 +
 +
Absorption is proportional to the path length through your sample. On a microtiter plate,
 +
this path length depends on how much sample you fill into your wells. If we know the exact inner dimensions
 +
of each well, we can easily calculate the path length for each volume. At least with the Greiner plate we
 +
tried, this works surprisingly well.
'''parameters'''
'''parameters'''
-
* well dimension: flat bottom 3.3 mm x 3.3 mm
 
* DNA extinction coefficient e: 50 [(ng * cm) / µl]
* DNA extinction coefficient e: 50 [(ng * cm) / µl]
 +
 +
* well dimension: flat bottom 3.3 mm x 3.3 mm
* path length in mm (d') or in cm (d):
* path length in mm (d') or in cm (d):

Revision as of 17:19, 28 May 2009

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Contents

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

Absorption is proportional to the path length through your sample. On a microtiter plate, this path length depends on how much sample you fill into your wells. If we know the exact inner dimensions of each well, we can easily calculate the path length for each volume. At least with the Greiner plate we tried, this works surprisingly well.

parameters

  • DNA extinction coefficient e: 50 [(ng * cm) / µl]
  • well dimension: flat bottom 3.3 mm x 3.3 mm
  • path length in mm (d') or in cm (d):
 d' = V / (3.32 * mm2)
 d = d' / 10

(V... volume per well in µl)

formula

 
  c = \frac{(A-A0) \times 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
  • e... extinction coefficient in ng*cm/µl

Procedure

  1. Pipette exact volume of DNA samples into 384 well plate
  2. measure 260 nm and 280 nm absorbance on plate reader
  3. Pipette sample back into storage plate
  4. 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.

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