Corum:DNA Quantification

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m (New page: ==Overview== DNA quantification using fluorescent quantifluore dye. ==Materials== * 200X stock quantifluore dye * 100 ng/μL stock DNA standard (salmon sperm) * DNA samples ==Procedure=...)
m (Procedure)
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#* 1/4000 = 2.5 μL 1/100 DNA + 97.5 μL TE
#* 1/4000 = 2.5 μL 1/100 DNA + 97.5 μL TE
# Prepare 2.5X quantifluore from 200X stock for N samples.
# Prepare 2.5X quantifluore from 200X stock for N samples.
-
#* V<sub>TE</sub> = [(80 + 40 × N) × 1.2] μL
+
#* V<sub>TE</sub> = 1.2 × [80 + 40 × N] μL = 1.2 × 40 × [N + 2] μL  
#* V<sub>200X</sub> = [V<sub>TE</sub> / (200/5 - 1 ) μL = V<sub>TE</sub> / 39] μL
#* V<sub>200X</sub> = [V<sub>TE</sub> / (200/5 - 1 ) μL = V<sub>TE</sub> / 39] μL
#* V<sub>2.5X</sub> = [V<sub>TE</sub> + V<sub>200X</sub>] μL
#* V<sub>2.5X</sub> = [V<sub>TE</sub> + V<sub>200X</sub>] μL

Revision as of 13:34, 26 September 2012

Contents

Overview

DNA quantification using fluorescent quantifluore dye.

Materials

  • 200X stock quantifluore dye
  • 100 ng/μL stock DNA standard (salmon sperm)
  • DNA samples

Procedure

  1. 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
  2. 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
  3. 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
  4. 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.)
  5. 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.
  6. Convert values to nM:
    • 1 nM = 106/(640 L) ng/μL (L = length of dsDNA; use 320 for ssDNA)

Notes

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Digital Signature

  • SC 17:44, 25 June 2012 (EDT):
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