Original reference in Wurst and Joyner chapter 2.
-Allow plates to grow confluent. If there are slow growers just keep feeding the fast ones until they all que up.
-Aspirate medium and wash twice with PBS.
-Apply lysis buffer according to the table below:
-Wrap dishes or plates in parafilm and enclose inside a humidified container. Incubate 3 hours-overnight at 37 degrees.
-If in 24-wells the precipitation can be done inside the dish, otherwise transfer the lysate to either a 15ml Falcon (35 or 60 mm) or a 50 ml Falcon (100 mm), using a cut blue tip or large bore pipet. Lysate is quite gooey.
|
dish |
volume lysis buffer, ml |
volume of TE to resuspend, ml |
|
24-well |
0.5 |
.050 |
|
35 mm |
2.0 |
.200 |
|
60 mm |
4.0 |
.400 |
|
100 mm |
10.0 |
1.0 |
|
100 mm Tris-HCl, pH 8.5 |
1.0 ml of 1 M Tris-HCl, pH 8.5 |
|
5mM EDTA |
0.1 ml of .5M EDTA |
|
0.2% SDS |
0.2 ml of 10% SDS |
|
200mM NaCl |
0.4 ml of 5M NaCl |
|
100µg/ml proteinase K |
0.050 ml of 20mg/ml proteinase K |
|
q.v. to 10.0 ml |
-Add an equal volume of Isopropanol to the dish or tube: just dump on top: do not mix vigorously
-Put some plastic wrap over the 24-wells and put lid back on. Weigh dish down with several lead pigs. Spin on an orbital shaker, starting at very low speed and gradually turning up until going the fastest possible without spilling the wells. Tubes are done on a nutator.
-Allow to rotate for at least 20 min at room temperature. A large white jelly fish will begin to appear at the interface of the alcohol with the lysate. The longer you rotate the tighter and easier to manage these jelly fishes will become.
-Using a pasteur pipet, carefully remove the supernatant leaving the jelly fish behind. Apply an equal volume of 70% ETOH and rotate for another 20 minutes at room temperature.
-Lift the precipitate out with a clean yellow tip (or blue tip if really big). Put the white mass into a tube with the appropriate amount of TE. Sometimes the precipitate sticks to the tip. Carefully scrape off on side of tube.
-Incubate tubes at 50-60 degrees for 30 minutes with lids open to evaporate trace ethanol.
-Store tubes in fridge overnight (at least)--good forever.
-Cut approximately 15 µl of DNA in a digestion with identical conditions to that used with the initial Southern screen (see above), but skip the RNAse (not needed). The DNA is very viscous and takes some effort to remove 15 µl exactly (don't worry if it's a little short). I normally run the entire reaction out on a 0.7%agarose/0.5x TBE gel as usual.
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