User:Elaine Haykin/Notebook/American University Chemistry Lab/2013/01/31

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(Cell Transformation)
(Cell Transformation)
Line 10: Line 10:
Note: Step 3 was skipped.
Note: Step 3 was skipped.
-
# Thaw the competent cells on ice.
 
-
# Gently mix the competent cells. Aliquot 100 μl of the competent cells
+
#1 Thaw the competent cells on ice.
 +
 
 +
#2 Gently mix the competent cells. Aliquot 100 μl of the competent cells
into the appropriate number of 14-ml BD Falcon polypropylene roundbottom
into the appropriate number of 14-ml BD Falcon polypropylene roundbottom
tubes. Prepare an additional 100-μl aliquot of cells for use as a
tubes. Prepare an additional 100-μl aliquot of cells for use as a
transformation control.
transformation control.
-
# Add 1.7 μl of the β-mercaptoethanol (β-ME) provided with this kit or a
+
#3 Add 1.7 μl of the β-mercaptoethanol (β-ME) provided with this kit or a
fresh 1:10 dilution of a 14.2 M stock solution of β-ME (diluted in
fresh 1:10 dilution of a 14.2 M stock solution of β-ME (diluted in
distilled water), to each polypropylene tube containing the competent
distilled water), to each polypropylene tube containing the competent

Revision as of 12:36, 31 January 2013

Project name Main project page

Cell Transformation

TRANSFORMATION PROTOCOL

Note: Step 3 was skipped.

  1. 1 Thaw the competent cells on ice.
  1. 2 Gently mix the competent cells. Aliquot 100 μl of the competent cells

into the appropriate number of 14-ml BD Falcon polypropylene roundbottom tubes. Prepare an additional 100-μl aliquot of cells for use as a transformation control.

  1. 3 Add 1.7 μl of the β-mercaptoethanol (β-ME) provided with this kit or a

fresh 1:10 dilution of a 14.2 M stock solution of β-ME (diluted in distilled water), to each polypropylene tube containing the competent cells, for a final concentration of 25 mM β-ME. Swirl the tubes gently.

  1. Incubate the reactions on ice for 10 minutes, swirling gently every

2 minutes.

  1. . Add 1–50 ng of ligated DNA to each transformation reaction and swirl

gently. For the control transformation reaction, add 1 μl of the pUC18 control plasmid to a separate 100-μl aliquot of the competent cells and swirl gently.

  1. Incubate the reactions on ice for 30 minutes.
  1. Preheat SOC medium (see Preparation of Media and Reagents) in a

42°C water bath for use in step 10.

  1. Heat-pulse each transformation reaction in a 42°C water bath for

45 seconds. The duration of the heat pulse is critical for optimal transformation efficiencies.

  1. Incubate the reactions on ice for 2 minutes.
  1. Add 0.9 ml of preheated (42°C) SOC medium to each transformation

reaction and incubate the reactions at 37°C for 1 hour with shaking at 225–250 rpm.


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