DNA quantification using fluorescent quantifluore dye.
- 200X stock quantifluore dye
- 100 ng/μL stock DNA standard (salmon sperm)
- DNA samples
- Prepare DNA standard curve:
- 1 ng/μL = 1 μL 100 ng/μL standard DNA + 99 μL TE
- 0.1 ng/μL = 10 μL 1 ng/μL standard DNA + 90 μL TE
- 0.01 ng/μL = 10 μL 0.1 ng/μL standard DNA + 90 μL TE
- Prepare DNA dilution samples.
- 1/100 = 1 μL sample DNA + 99 μL TE
- 1/2000 = 5 μL 1/100 DNA + 95 μL TE
- 1/4000 = 2.5 μL 1/100 DNA + 97.5 μL TE
- Prepare 2.5X quantifluore from 200X stock for N samples.
- VTE = 1.2 × [80 + 40 × N] μL = 1.2 × 40 × [N + 2] μL
- V200X = [VTE / (200/5 - 1 ) μL = VTE / 39] μL
- V2.5X = [VTE + V200X] μL
- Aliquot 80 v blank TE, standard DNA curve, and diluted DNA samples onto a 96 well plate. Add 20 μL 2.5X quantifluore per well. (Note: final quantifluore concentration is 0.5X, not 1X.)
- With the plate reader, shake double-orbital fast 30 s, pause 10 min, shake double-orbital fast 30 s again, and then read end-point fluorescence at 485/528 nm. The plate reader returns the data in ng/μL concentration values.
- Convert values to nM:
- 1 nM = 106/(640 L) ng/μL (L = length of dsDNA; use 320 for ssDNA)
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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- SC 17:44, 25 June 2012 (EDT):