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

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(Cell Transformation)
(Cell Transformation)
 
Line 12: Line 12:
  
 
# Thaw the competent cells on ice.
 
# Thaw the competent cells on ice.
 
+
# 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.
# Gently mix the competent cells. Aliquot 100 μl of the competent cells
+
# 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.
into the appropriate number of 14-ml BD Falcon polypropylene roundbottom
+
# Incubate the reactions on ice for 10 minutes, swirling gently every 2 minutes.
tubes. Prepare an additional 100-μl aliquot of cells for use as a
+
# 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.
transformation control.
 
 
 
# 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.
 
 
 
# Incubate the reactions on ice for 10 minutes, swirling gently every
 
2 minutes.
 
 
 
#. 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.
 
 
 
 
# Incubate the reactions on ice for 30 minutes.
 
# Incubate the reactions on ice for 30 minutes.
 
+
# Preheat SOC medium (see Preparation of Media and Reagents) in a 42°C water bath for use in step 10.
# Preheat SOC medium (see Preparation of Media and Reagents) in a
+
# 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.
42°C water bath for use in step 10.
 
 
 
# 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.
 
 
 
 
# Incubate the reactions on ice for 2 minutes.
 
# Incubate the reactions on ice for 2 minutes.
 
+
# 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.
# 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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Latest revision as of 10:45, 31 January 2013

Owwnotebook icon.png 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.
  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.
  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.
  4. Incubate the reactions on ice for 10 minutes, swirling gently every 2 minutes.
  5. 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.
  6. Incubate the reactions on ice for 30 minutes.
  7. Preheat SOC medium (see Preparation of Media and Reagents) in a 42°C water bath for use in step 10.
  8. 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.
  9. Incubate the reactions on ice for 2 minutes.
  10. 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.