Maxiprep of Plasmid DNA
For obtain human liver cell cDNA library in YEp552 vector from E.coli
User guide of “ PureLink® HiPure Plasmid Filter Purification Kits For Midi and Maxi preparation of Plasmid DNA “ written below:
- The HiPure technology, based on anion-exchange chromatography, uses a patented resin composed of small particles with a uniform pore size.
-Column equilibrated with EQ1. E. coli cells are harvested with a centrifuge, then resuspended in Resuspension Buffer (R3) with RNase A, and lysed with Lysis Buffer (L7, SDS/alkaline lysis). Precipitation Buffer (N3 P.Ac) is then added to the lysate. The lysate is poured into a pre-packed anion-exchange column fitted with the Filtration Cartridge unit. In one simple combined step, the lysate is clarified, and the negatively charged phosphates of the DNA backbone interact with the positive charges on the surface of the anionexchange resin. The temperature, salt concentration, and pH of the solutions are optimized for efficient binding of DNA. A single column wash under moderate salt conditions using Wash Buffer (W8) removes RNA, proteins, carbohydrates and other impurities while the plasmid DNA remains bound to the resin. The plasmid DNA is eluted under high salt conditions with the Elution Buffer (Tris-HCl +NaCl). The eluted DNA is desalted and concentrated by alcohol precipitation. TE buffer used to resuspend the DNA pellet.
Isopropanol Precipitation: The salts neutralize the negative charge of the negatively charged phosphate in DNA and the isopropanol /ethanol removes the hydration shell of H2O molecules around the phosphate.
DNA Yield Measure the DNA concentration using UV absorbance at 260 nm or Qubit® DNA Assay Kits. UV Absorbance 1. Dilute the DNA in 10 mM Tris-HCl, pH 7.5. Mix well. Measure the absorbance at 260 nm (A260) of the dilution in a spectrophotometer (using a cuvette with an optical path length of 1 cm) blanked against 10 mM Tris-HCl pH 7.5 2. Calculate the concentration of DNA using the formula: DNA (g/mL) = A260 50 × dilution factor For DNA, A260 = 1 for a 50 g/mL solution measured in a cuvette with an optical path length of 1 cm.
