Difference between revisions of "Bacterial Transformation for Bio113"

Revision as of 20:03, 18 July 2017

Thaw the competent cells on ice for 6 minutes. Each tube contains 50 µL of cells.

Add all 10 µL of your GGA ligation mixture to the thawed cells. Very gently mix the DNA and cells and return the cells to ice ASAP.

Add 60 µL SOC media with no antibiotic to your transformed cells. Spread cells onto LB plates containing ampicillin.

You're done already!!

@@ Line 2: / Line 2: @@
 == Transforming DNA after GGA Ligation ==
-# Thaw the competent cells on ice for 6 minutes. Each tube contains 50 µL of cells. You can use these straight. Diluting up to 10 fold will work for ligations as well as direct transformations of minipreps.
+# Thaw the competent cells on ice for 6 minutes. Each tube contains 50 µL of cells.
-) Very gently, aliquot cells into smaller volumes (we have used as low as 20 µL of cells with 5 µL of ligation) using pre-chilled microfuge tubes.
+# Add all 10 µL of your GGA ligation mixture to the thawed cells. Very gently mix the DNA and cells and return the cells to ice ASAP.
-) Add 1 - 5 µL of ligation mixture (can go as high as 10 µL for larger volumes of cells).
+# Incubate on ice for 5 minutes.
-) Incubate on ice for 5 minutes.
+# Add 60 µL SOC media with no antibiotic to your transformed cells. Spread cells onto LB plates containing ampicillin.
-) Add SOC media with no antibiotic to a final volume of 60 - 100 µL. Spread cells onto plates containing ampicillin*. You must let this sit for 30 minutes in the liquid at 37 C before plating if the antibiotic is not ampicillin.
 You're done already!!