Exercise 14.2 - Purification of DNA
- Spooled DNA from Exercise 14.1
- SSC buffer
- Pancreatic Ribonuclease A (100 µg/ml)
- Sodium lauryl sulfate (SLS)
- Sodium perchlorate
- Chloroform:isoamyl alcohol (24:1)
- 95% and 70% (v/v) ethanol
- Refrigerated centrifuge, rotor and tubes
- Decant off the alcohol, and dissolve the extracted DNA in 30
ml. of diluted SSC buffer (0.1 X SSC) in a 125 ml. erlenmeyer
flask. This will require some time, as polymerized DNA dissolves
slowly. Gentle swirling of the material will help.
- Add pancreatic ribonuclease A to a final concentration of
100 micrograms/ml and agitate slowly at 37° C for 1
- Add pronase to a final concentration of 50 micrograms/ml and
again agitate slowly at 37° C for another hour.
- Add sodium lauryl sulfate (SLS) 2
to make a 1% concentration (w/v) and sodium perchlorate to a
final concentration of 1 M. Agitate for 30 minutes at room
- Extract the solution with chloroform:isoamyl alcohol (24:1
v/v) by adding an equal volume and shaking virogously for at
least 15 minutes.
- Place the solution into appropriate centrifuge tubes and
centrifuge for 5 minutes at 800 xg and 4° C.
- Remove the upper aqueous phase, add 2.3 volumes of 95% cold
ethanol and respool the DNA from this solution onto a glass rod.
- Wash the spooled DNA twice with cold 70% ethanol and store
for future analysis.
to Table of Contents
Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org