Difference between revisions of "Bacterial Transformation for Bio113"

Latest revision as of 17:49, 2 September 2022

Thaw the competent cells on ice for 6 minutes. Each tube contains 50 µL of E. coli, JM109 cells.
Add all 50 µL of your E. coli cells to the GGA ligation mixture. Very gently mix the DNA and cells and return the cells to ice ASAP.
Incubate on ice for 5 minutes.
Spread cells onto LB plates containing ampicillin.

You're done already!!

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 == 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 ''E. coli'', JM109 cells.
+# Add all 50 µL of your ''E. coli'' cells to the GGA ligation mixture. Very gently mix the DNA and cells and return the cells to ice ASAP.
-) 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.
+# Incubate on ice for 5 minutes.
+# Spread cells onto LB plates containing ampicillin.
-) 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.
-) 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!!