EXERCISE 12

 

 

DNA Isolation and Gel Electrophoresis

(adapted from Sambrook, Fritsch and Maniatis. Molecular Cloning, 1989)

 

Supplies Required:

            Phenol:Chloroform (25:25) or Phenol:Chloroform:Isoamyl Alcohol (25:24:1)

            Chloroform (not mixed with phenol)

            Ice cold ethanol

            Microfuge tubes

            Micropipettes

            Pipette tips

 

Protocol:

 

  1. Prepare sample from which DNA will be extracted. (i.e. Spin down bacterial cells, homogenize or blend sample).
  2. Add 500ml (or an equal volume) of Phenol:Chloroform to the prepared sample.
  3. Centrifuge samples at 12,000g for 1 min or at 1600g for 5 minutes to separate sample into two phases (organic and aqueous phases).
    1. You should be able to see some protein at the interface between the two layers.
  4. Transfer the top layer (aqueous phase) to a new tube and discard the protein-containing organic phase (bottom layer).
    1. Make sure not to transfer any protein (a.k.a. the Ôwhite stuffÕ at the aqueous:organic phase junction.
    2. Make sure to note how much liquid you are taking from the aqueous layer so you can perform STEP 6!!
  5. Repeat steps 1-4 until you remove all protein from the aqueous:organic phase junction.
  6. Add an equal volume of Chloroform (without Phenol) to the aqueous layer.
    1. If you have 200ml of aqueous layer in your tube, add 200ml of Chloroform
  7. Centrifuge as in STEP 3.
  8. Transfer the TOP layer into a fresh tube.
    1. Note Volume for STEP 9!!
  9. Add an equal volume of ICE COLD Ethanol
    1. Let stand for 20 minutes.
  10. Centrifuge as in STEP 3 to pellet DNA.
  11. Decant liquid
    1. BE SURE NOT TO DISTURB THE PELLET
  12. Add 50ml H2O to sample to reconstitute DNA