User:Elaine Haykin/Notebook/American University Chemistry Lab/2013/01/31: Difference between revisions
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==Cell Transformation== | ==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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Revision as of 10:25, 31 January 2013
 Project name
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<html><img src="/images/9/94/Report.png" border="0" /></html> Main project page |
Cell Transformation
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. | |
