Difference between revisions of "Transformation via Electroporation"
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== Materials Needed == | == Materials Needed == | ||
| + | -Electroporation apparatus | ||
| + | |||
| + | -1 mm electroporation cuvette | ||
| + | |||
| + | -micropipette | ||
| + | |||
| + | -LB broth | ||
| + | |||
| + | -Electrocompetent cells | ||
| + | |||
| + | -DNA | ||
| + | |||
| + | -microfuge tubes | ||
| + | |||
| + | -ice | ||
| + | |||
| + | -kimwipes | ||
| + | |||
| + | -LB or NBY agar plates | ||
| + | |||
| + | -28 C incubator | ||
== Protocol == | == Protocol == | ||
| − | -Set the electroporation apparatus to 1 | + | -Set the electroporation apparatus to 1.8 kV, 25 microfarads, and the pulse controller to 200 omega |
-Add 1.5 microliters of DNA to be transformed to 40 microliters of electrocompetent cells on ice and mix | -Add 1.5 microliters of DNA to be transformed to 40 microliters of electrocompetent cells on ice and mix | ||
| Line 20: | Line 41: | ||
-Remove the cuvette and ad 1 mL LB broth to the cuvette | -Remove the cuvette and ad 1 mL LB broth to the cuvette | ||
| − | -Transfer the broth and cells to a sterile tube and incubate for 2 hours at 28 C with shaking | + | -Transfer the broth and cells to a sterile microfuge tube and incubate for 2 hours at 28 C with shaking |
| − | -Place aliquots of the mixture on LB or NBY agar plates with the appropriate antibiotics for the cells | + | -Place aliquots of the mixture on LB or NBY agar plates with the appropriate antibiotics for selection the cells |
-Incubate at 28 C for 2 days | -Incubate at 28 C for 2 days | ||
Latest revision as of 14:21, 20 July 2015
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Contents
Purpose
This protocol is used to transform electrocompetent cells with a plasmid.
The electrocompetent cells are made using the protocol found on Preparation of Competent Cells for Electroporation.
Materials Needed
-Electroporation apparatus
-1 mm electroporation cuvette
-micropipette
-LB broth
-Electrocompetent cells
-DNA
-microfuge tubes
-ice
-kimwipes
-LB or NBY agar plates
-28 C incubator
Protocol
-Set the electroporation apparatus to 1.8 kV, 25 microfarads, and the pulse controller to 200 omega
-Add 1.5 microliters of DNA to be transformed to 40 microliters of electrocompetent cells on ice and mix
-Transfer the DNA and cells to a pre-chilled 1 mm electroporation cuvette
-Wipe the cuvette with a kimwipe and place it into the sample chamber
-Deliver a pulse by pushing in charge button
-Remove the cuvette and ad 1 mL LB broth to the cuvette
-Transfer the broth and cells to a sterile microfuge tube and incubate for 2 hours at 28 C with shaking
-Place aliquots of the mixture on LB or NBY agar plates with the appropriate antibiotics for selection the cells
-Incubate at 28 C for 2 days
Notes
Papers where this or a similar method has been used
Related Ontology Terms
References
See Help:References for how to manage references in OMPwiki.
