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DNA ISOLATION AND PLASMID PURIFICATION

> Phenol extraction and ethanol precipitation.

   

> Large Scale Plasmid Prep for 100 ML Superbroth Cultures

   

> Plasmid Miniscreen Protocol (Boil Prep)

   

> Alkaline Lysis Miniprep

   

> Alkaline Lysis Miniprep Modified 1

   

> Alkaline Lysis Miniprep Modified 2

   

> Amplification of plasmids with chloramphenicol

   

> Single-Strand DNA Prep. Large Scale

    > Titre of Bacteriophage
  > Large Scale Bacteriophage Prep

Phenol Extraction and Ethanol Precipitation

Get a printable version of this protocol (requires Adobe Acrobat

  1. Add an equal volume of TE saturated phenol to the solution containing the DNA to be purified. Safety Notes 
  2. Vortex 10 sec. and spin in microfuge for 30 sec.
  3. Suck off the organic phase (yellow). The organic phase (phenol solution) is usually on the bottom however, high salt concentrations occasionally result in the inversion of the layers. Either way, you want to get rid of the yellow one.
  4. Add 1/10th volume of ammonium acetate (5M), finger vortex and add 2.5 vols of ethanol or 0.6 vols isopropanol. Isopropanol is good for relativiely large pieces of DNA (the size of a plasmid) as less RNA and free nucleotides are precipitated. Since isopropanol is more deleterious to enzymes etc. always wash the DNA pellet with 70% ethanol.  Because residual isopropanol is more of a problem than residual ethanol I usually use ethanol for precipitating unless isopropanol is specified (ie plasmid preps). Contrary to popular misconception there is no advantage to cooling (or freezing) after adding ethanol or isopropanol. Room temp. Is fine and may even be better. Safety Notes
  5. Spin 10 min in microfuge, suck off sup carefully, wash pellet by addition of 70% ethanol (volume equal to the original total) and respin 5 minutes, suck dry carefully with a pulled pipette, air dry at 37C for a few minutes or with speed vac and dissolve in TE (volume depends on the next step). After the wash the DNA pellet does not stick very well to the side of the tube so be careful. If the DNA is small (less than 200 bases) wash with 95% ethanol.

Safety Notes:

Phenol is nasty stuff. It denatures proteins so that is what it will do to the proteins that make up the skin on your hands and arms, etc. Wear gloves, labcoat and safety glasses--NO EXCEPTIONS.  
Ethanol and isopropanol are flammable and should be stored in the flammable liquids cupboard when not in use. 
Read the MSDS for these compounds and don't be careless.