In general, the method may be outlined as follows.
The cells are first disrupted, the cell debris and
protein are removed by denaturation and centrifugation, the RNA is then removed by selective
precipitation of the DNA with ethanol. Degradation by DNAase and divalent metal ion contamination is
prevented by the presence of chelating agents and by the action of sodium dodecyl sulphate. The
products obtained, although they may not reflect the in vivo molecular weight, have molecular weights
in excess of 10 x 106 g/mol.
Procedure
7m1 Naclow
Week 1: Extraction of DNA: 1-2g M. luteus.
Prepare a suspension of 1g Micrococcus lysodeikticus cells in 25 mL of saline EDTA. Place the cells in a
glass stoppered flask (use a powder funnel) and after adding the saline EDTA, stir well to make a
homogeneous slurry of the cells for incubation. Add 1 mL of lysozyme solution, mix and incubate in a
water bath at 37°C with frequent swirling for 20-25 minutes. The suspension should appear more
viscous due to cell lysis.
As soon as the cells have lysed, add 2.0 mL sodium dodecyl sulphate, mix, and heat the mixture in a
water bath at 60°C for 10 minutes. Cool to room temperature.
NaCIO4 is added to a final concentration of 1 M to the lysed suspension. (Calculate the number of mL of
7mL NaClO4 required and add this volume.) To the total volume now in the flask, add 35m an equal
volume of chloroform: isoamyl alcohol 35 mL. Stopper and shake, first vigorously by hand (WEARING
GLOVES), and then on a shaker. Continue shaking for 20 minutes, making sure the emulsion is complete.
A creamy emulsion should result. Centrifuge this at 12,000 rpm in the high speed centrifuge for 6
minutes. Three layers will result. The upper aqueous phase contains the nucleic acids. Using a Pasteur
pipette and rubber bulb, remove this upper layer carefully and place it in a 100 mL graduated cylinder.
Measure the volume.
Gently layer about 2 volumes (i.e. 2 times the volume of the upper aq. layer) of ethanol on the aqueous
phase. This will precipitate the nucleic acids. Stir the interface gently with a long coarse stirring rod and
the nucleic acids will "spool" out on the rod as a threadlike precipitate. Collect in this way as much as
possible of the nucleic acids.
The precipitate should be drained free of excess alcohol by pressing the spooled rod against the vessel
wall. Transfer the DNA to about 10-15 mL dilute saline-citrate to a boiling tube (record the actual
volume used) and gently remove it from the stirring rod by swirling the rod back and forth. The solution
should be slowly pipetted with a Pasteur pipette until dispersion is complete. (Lumps may be recognized
by adhering air bubbles when the solution is shaken.)
Week 2: Characterization:
Make a careful and accurately noted dilution of the DNA sample so that the absorption at 260 nm is
about 0.5. Try diluting 2.5 mL of stock DNA solution with 7.5 mL of water. Using a UV/quartz cuvette,
zero the spectrophotometer at 360 nm first using water.
Next, record the absorbance of your diluted DNA sample at 260 nm and 280 nm and determine the DNA
concentration using the nomograph (handout). Run a spectrum from 360-200 nm. Bring your flash drive
to download spectral data.
Put the diluted sample back in a test tube and heat it in a boiling water bath for 10-15 minutes. Cool the
sample quickly by placing it immediately in an ice bath. Rerun the spectrum on the denatured sample
and re-determine the absorbance at 260 nm. Compare with the spectrum and absorbance value
obtained earlier. Explain any differences noted
Discussion
Interpreted results in the isolation procedure, explaining how each experimental step allowed for
isolation/purification of DNA
Identified any problems and errors that could result, and provided suggestions for improvement
Determined if isolation was successful, if DNA pure and provided evidence.
· Discussion on denatured DNA and how it compared to native
