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Quantitation of DNA for sequencing using the TKO100 fluorometer

Quantitation of DNA for sequencing using the TKO100 fluorometer

  1. Turn on the machine and allow it to warm up for 15-30 minutes; make sure the machine and all solutions are at RT.
  2. Place 2 ml of the TNE-dye solution in the cuvette and place the cuvette in the fluorometer.
  3. Zero the machine using the zero dial on the fluorometer.
  4. Add 1 ul of your DNA standard. For plasmid DNA use the 0.2mg/L standard that comes with the ABI DNA sequencing kit. Mix the cuvette by hand and place back in the fluorometer.
  5. Adjust the scale button until it reads 100 units.
  6. Remove the cuvette and empty its contents. Repeat steps 2-5 several times until you get consistent readings. An occasional bad reading is often the result of pipetting error; note that not all the Hancock lab P2 pipetters deliver consistent volumes and this results in readings that can be interpreted as ‘drift’ by the fluorometer. Drift of +/- 5 units can be ignored (i.e. readings of 95 to 105).
  7. Repeat steps 2-5 using your own DNA samples. Record the fluorescence units.
  8. DNA quantity is calculated as follows: 2 X (your fluorescence units) = DNA concentration in nanograms per microliter.
  9. If your DNA sequence is less than 70 ng/ul throw it out and make new template; you will need a significantly higher concentration of DNA if you are using plasmids larger that 10 kb (>150 ng/ul). Using the Qiagen preps and high copy plasmids you should expect your concentration to be in the range of 80-200 ng/ul. I have found it difficult to get consistent DNA sequencing results using Qiagen prep DNA at concentration less than 70 ng/ul.

Solutions:

10X TNE

For 500 mL solution.

Tris

6.05g

Na2-EDTA

1.85g

NaCl

29.2g

dissolve above into 400 ml ddH2O and pH to 7.4 with concentrated Hl
Make to 500 ml

Concentrated dye stock solution

Hoechst 33258 1 mg/ml in ddH2O; -store this in the freezer

Working dye solution

  1. Add 10 ul of concentrated dye solution to 100 ml 1X TNE
  2. Pass the solution through a 0.22-0.5 um filter.
  3. Store this solution in a dark bottle at RT for up to 3 months. Note that this solution will go off and you will get inconsistent, or no results at all.