Bacterial Transformation for Bio113 - Revision history

Macampbell: /* Transforming DNA after GGA Ligation */

2022-09-02T17:49:12Z

‎Transforming DNA after GGA Ligation

Macampbell at 19:32, 23 October 2018

2018-10-23T19:32:18Z

Macampbell at 19:21, 23 October 2018

2018-10-23T19:21:37Z

Macampbell at 13:06, 6 August 2018

2018-08-06T13:06:08Z

Macampbell at 20:03, 18 July 2017

2017-07-18T20:03:39Z

Macampbell at 20:03, 18 July 2017

2017-07-18T20:03:19Z

Macampbell: Created page with " == 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..."

2017-07-18T19:20:25Z

Created page with " == 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..."

New page

== 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.

2) 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.

3) Add 1 - 5 µL of ligation mixture (can go as high as 10 µL for larger volumes of cells).

4) Incubate on ice for 5 minutes.

5) 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!!

@@ Line 5: / Line 5: @@
 # 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.
-# Add 30 µL SOC media with no antibiotic to your transformed cells. Spread cells onto LB plates containing ampicillin.
+# Spread cells onto LB plates containing ampicillin.
 You're done already!!

@@ Line 5: / Line 5: @@
 # 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.
-# Add 60 µL SOC media with no antibiotic to your transformed cells. Spread cells onto LB plates containing ampicillin.
+# Add 30 µL SOC media with no antibiotic to your transformed cells. Spread cells onto LB plates containing ampicillin.
 You're done already!!

@@ Line 3: / Line 3: @@
 # Thaw the competent cells on ice for 6 minutes. Each tube contains 50 µL of ''E. coli'', JM109 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 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.
 # 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.
+# Thaw the competent cells on ice for 6 minutes. Each tube contains 50 µL of ''E. coli'', JM109 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.
 # 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.
 You're done already!!

@@ Line 3: / Line 3: @@
 # 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.
 # 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.
 You're done already!!