Bologna University/Transformation

< Bologna University(Difference between revisions)

Latest revision as of 09:44, 25 October 2007

1. Thaw the competent cells in ice (do not refreeze).

2. Dispense 100μl of cells into microfuge tubes on ice.

3. Add 0.1-0.3μg of plasmidic DNA or the respective amount of the ligation reaction.

4. Keep on ice for 30min.

5. Heat at 42 °C for 60sec without agitation.

6. Keep on ice for 2min.

7. Add 0.9ml of LB medium at room temperature.

8. Incubate at 37 °C for 1hr with agitation.

9. Pellet the cells and discard most of supernatant, leaving about 100μl.

10. Streak on plates containing appropriate antibiotics.

11. Incubate the plates overnight at 37 °C.

@@ Line 1: / Line 1: @@
 ::1. Thaw the competent cells in ice (do not refreeze).
-::2. Dispense 100 μl of cells into microfuge tubes on ice.
+::2. Dispense 100μl of cells into microfuge tubes on ice.
-::3. Add 0.1-0.3 μg of plasmidic DNA or the respective amount of the ligation reaction.
+::3. Add 0.1-0.3μg of plasmidic DNA or the respective amount of the ligation reaction.
-::4. Keep on ice for 30 min.
+::4. Keep on ice for 30min.
-::5. Heat at 42 °C for 60 seconds without agitation.
+::5. Heat at 42 °C for 60sec without agitation.
-::6. Keep on ice for 2 minutes.
+::6. Keep on ice for 2min.
-::7. Add 0.9 ml of LB medium at room temperature.
+::7. Add 0.9ml of LB medium at room temperature.
-::8. Incubate at 37 °C for 1 hr with agitation.
+::8. Incubate at 37 °C for 1hr with agitation.
-::9. Pellet the cells and discard most of supernatant, leaving about 100 μl.
+::9. Pellet the cells and discard most of supernatant, leaving about 100μl.
 ::10. Streak on plates containing appropriate antibiotics.
 ::11. Incubate the plates overnight at 37 °C.