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

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# Thaw the competent cells on ice.
# Thaw the competent cells on ice.


2. Gently mix the competent cells. Aliquot 100 μl of the competent cells
# 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.


3. Add 1.7 μl of the β-mercaptoethanol (β-ME) provided with this kit or a
# 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
cells, for a final concentration of 25 mM β-ME. Swirl the tubes gently.
cells, for a final concentration of 25 mM β-ME. Swirl the tubes gently.
4. Incubate the reactions on ice for 10 minutes, swirling gently every
 
# Incubate the reactions on ice for 10 minutes, swirling gently every
2 minutes.
2 minutes.


5. Add 1–50 ng of ligated DNA to each transformation reaction and swirl
#. 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
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
control plasmid to a separate 100-μl aliquot of the competent cells and
swirl gently.
swirl gently.


6. Incubate the reactions on ice for 30 minutes.
# Incubate the reactions on ice for 30 minutes.


7. Preheat SOC medium (see Preparation of Media and Reagents) in a
# Preheat SOC medium (see Preparation of Media and Reagents) in a
42°C water bath for use in step 10.
42°C water bath for use in step 10.


8. Heat-pulse each transformation reaction in a 42°C water bath for
# Heat-pulse each transformation reaction in a 42°C water bath for
45 seconds. The duration of the heat pulse is critical for optimal
45 seconds. The duration of the heat pulse is critical for optimal
transformation efficiencies.
transformation efficiencies.


9. Incubate the reactions on ice for 2 minutes.
# Incubate the reactions on ice for 2 minutes.


10. Add 0.9 ml of preheated (42°C) SOC medium to each transformation
# 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
reaction and incubate the reactions at 37°C for 1 hour with shaking at
225–250 rpm.
225–250 rpm.

Revision as of 10:35, 31 January 2013

Project name <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page

Cell Transformation

TRANSFORMATION PROTOCOL

Note: Step 3 was skipped.

  1. Thaw the competent cells on ice.
  1. 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. 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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