Cumbers:protocols/picogreen quantIT

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Cumbers mutterings

Many of these pages are rough notes and protocols that I'm working on. I'm trying to publish as many of my ideas on oww to promote an open type of science, for discussion and collaboration. Please feel free to browse and read them. If you find any errors or anything of interest then please comment in the discussion page, or e-mail me your thoughts.

  • Follow the Quant-it protocol for DNA (and RNA) quantification.
  • Make up standard measurements along with your samples.
  • If unsure of your scale (if you have a lot or a little) then do 3 concentrations of your sample to check, one at 100% concentration, one at 1:10 other at 1:100
  • First you are making a 1:10 (add 1ul DNA to 9ul TE to make 1:10), then you are taking 1 uL of this 1:10 solution and adding it to 99 uL. So that is 1:100 of the 1:10 so the solution where you took 1 uL of this 1:10 solution and added to 99ul TE is 1:1000 of the original DNA concentration (1:10 * 1:100).
  • Do 2 of TE or whatever you are resuspending in to subtract this background from your results.
  • set up the plate reader and computer.
  • Add the fluorescent reagent (make up enough: 100 assays = 20 ml HS buffer) - 200ul added to each plate, make sure you have enough and careful when pipetting, keep track of which hole you want to add it to.
  • read the plate
   1   2   3         4  
A  10  10  1C        1S
B  8   8   1C(1:10)  1S(1:10)
C  6   6   1C(1:10)  1S(1:10)
D  4   4   1C(1:10)  1S(1:10)
E  2   2   1C(1:100) 1S(1:100)
F  1   1   1C(1:100) 1S(1:100)
G  0.5 0.5 1C(1:100) 1S(1:100)
H  0   0   TE        TE