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

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(Cell Transformation)
(Cell Transformation)
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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.
2. Gently mix the competent cells. Aliquot 100 μl of the competent cells
2. Gently mix the competent cells. Aliquot 100 μl of the competent cells

Revision as of 12:31, 31 January 2013

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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