User:Elaine Haykin/Notebook/American University Chemistry Lab/2013/01/31: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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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 tubes. Prepare an additional 100-μl aliquot of cells for use as a 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 distilled water), to each polypropylene tube containing the competent cells, for a final concentration of 25 mM β-ME. Swirl the tubes gently. | |||
into the appropriate number of 14-ml BD Falcon polypropylene roundbottom | # Incubate the reactions on ice for 10 minutes, swirling gently every 2 minutes. | ||
tubes. Prepare an additional 100-μl aliquot of cells for use as a | # 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. | ||
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 | |||
distilled water), to each polypropylene tube containing the competent | |||
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. | |||
# | |||
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. | |||
# Incubate the reactions on ice for 30 minutes. | # 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. | |||
# Preheat SOC medium (see Preparation of Media and Reagents) in a | # 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. | ||
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 | |||
transformation efficiencies. | |||
# Incubate the reactions on ice for 2 minutes. | # 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 225–250 rpm. | |||
# 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. | |||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> |
Latest revision as of 22:24, 26 September 2017
Project name | Main project page |
Cell TransformationTRANSFORMATION PROTOCOL Note: Step 3 was skipped.
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