Pelling:Protocols/Plasmid DNA Isolation

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Plasmid DNA Isolation From 3mL DH5α Cultures

We use the Qiagen Spin MiniPrep kit and a protocol can be found here

DNA Quantification

  1. Turn on UV Spectrophotometer.
  2. Mode “1” for DNA quantification.
  3. Need to read absorbance at 260nm, 280nm, and the ratio A260/A280.
  4. Set molar absorptivity to ε = 50, path length should be set already.
  5. dilution factor: 1:50.
  6. Dilute 1:50 in the amounts as follows: 8μL DNA with 392μL dH2O (400μL total volume).
  7. Use 400μL dH2O as a blank/reference on the machine.
  8. Calculate the DNA concentration for each solution.
  9. Conc. DNA = A260 x dilution factor (ie 50) x 50μg/μL (should be given by machine).
  10. Purity of DNA: Want the A260/A280 ratio about 1.8-2.0.
  11. Calculate the volume of DNA necessary for 1μg DNA.
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