Prbbbb:dna measurement on plate v1

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(Calculation)
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* DNA extinction coefficient e: 50 [(ng * cm) / µl]
* DNA extinction coefficient e: 50 [(ng * cm) / µl]
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* path length in mm / in cm:
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* path length in mm (d') or in cm (d):
   d' = V / (3.3^2 * mm^2)
   d' = V / (3.3^2 * mm^2)
   d  = d'/10  
   d  = d'/10  
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'''formula'''
'''formula'''
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  c = (A-A0 * e) / d
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<math>
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c = ((A-A0) * e) / d
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</math>
* c... DNA concentration in ng / µl
* c... DNA concentration in ng / µl

Revision as of 17:02, 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

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

c = ((AA0) * 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

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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