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

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==Cell Transformation==
==Cell Transformation==
-
* Insert content here...
+
*TRANSFORMATION PROTOCOL
 +
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.
 +
 +
Note: Step 3 was skipped.
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Revision as of 13:25, 31 January 2013

Project name Main project page

Cell Transformation

  • TRANSFORMATION PROTOCOL

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.

Note: Step 3 was skipped.


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