User:Elaine Haykin/Notebook/American University Chemistry Lab/2013/01/31: Difference between revisions
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# 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 | into the appropriate number of 14-ml BD Falcon polypropylene roundbottom | ||
tubes. Prepare an additional 100-μl aliquot of cells for use as a | tubes. Prepare an additional 100-μl aliquot of cells for use as a | ||
transformation control. | 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 | 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 | distilled water), to each polypropylene tube containing the competent | ||
cells, for a final concentration of 25 mM β-ME. Swirl the tubes gently. | 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. | 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 | 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 | control plasmid to a separate 100-μl aliquot of the competent cells and | ||
swirl gently. | swirl gently. | ||
# 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. | 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 | 45 seconds. The duration of the heat pulse is critical for optimal | ||
transformation efficiencies. | transformation efficiencies. | ||
# 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 | reaction and incubate the reactions at 37°C for 1 hour with shaking at | ||
225–250 rpm. | 225–250 rpm. |
Revision as of 10:35, 31 January 2013
Project name | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page |
Cell TransformationTRANSFORMATION PROTOCOL Note: Step 3 was skipped.
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.
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.
2 minutes.
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.
42°C water bath for use in step 10.
45 seconds. The duration of the heat pulse is critical for optimal transformation efficiencies.
reaction and incubate the reactions at 37°C for 1 hour with shaking at 225–250 rpm. |