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

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(Cell Transformation)
Current revision (13:45, 31 January 2013) (view source)
(Cell Transformation)
 
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==Cell Transformation==
==Cell Transformation==
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*TRANSFORMATION PROTOCOL
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TRANSFORMATION PROTOCOL
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Note: Step 3 was skipped.
Note: Step 3 was skipped.
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1. Thaw the competent cells on ice.
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# Thaw the competent cells on ice.
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# 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.
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2. Gently mix the competent cells. Aliquot 100 μl of the competent cells
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# 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.
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into the appropriate number of 14-ml BD Falcon polypropylene roundbottom
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# Incubate the reactions on ice for 10 minutes, swirling gently every 2 minutes.
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tubes. Prepare an additional 100-μl aliquot of cells for use as a
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# 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.
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transformation control.
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# Incubate the reactions on ice for 30 minutes.
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# Preheat SOC medium (see Preparation of Media and Reagents) in a 42°C water bath for use in step 10.
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3. Add 1.7 μl of the β-mercaptoethanol (β-ME) provided with this kit or a
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# 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.
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fresh 1:10 dilution of a 14.2 M stock solution of β-ME (diluted in
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# Incubate the reactions on ice for 2 minutes.
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distilled water), to each polypropylene tube containing the competent
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# 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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cells, for a final concentration of 25 mM β-ME. Swirl the tubes gently.
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-
 
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4. Incubate the reactions on ice for 10 minutes, swirling gently every
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2 minutes.
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5. Add 1–50 ng of ligated DNA to each transformation reaction and swirl
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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.
+
-
 
+
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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.
+
-
 
+
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10. Add 0.9 ml of preheated (42°C) SOC medium to each transformation
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reaction and incubate the reactions at 37°C for 1 hour with shaking at
+
-
225–250 rpm.
+
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Current revision

Project name 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.


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