Plasmid DNA Isolation From 3mL DH5α Cultures
We use the Qiagen Spin MiniPrep kit and a protocol can be found here
- Turn on UV Spectrophotometer.
- Mode “1” for DNA quantification.
- Need to read absorbance at 260nm, 280nm, and the ratio A260/A280.
- Set molar absorptivity to ε = 50, path length should be set already.
- dilution factor: 1:50.
- Dilute 1:50 in the amounts as follows: 8μL DNA with 392μL dH2O (400μL total volume).
- Use 400μL dH2O as a blank/reference on the machine.
- Calculate the DNA concentration for each solution.
- Conc. DNA = A260 x dilution factor (ie 50) x 50μg/μL (should be given by machine).
- Purity of DNA: Want the A260/A280 ratio about 1.8-2.0.
- Calculate the volume of DNA necessary for 1μg DNA.