Difference between revisions of "Preparation of Competent Cells for Electroporation"
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== Purpose == | == Purpose == | ||
| + | This protocol is used for preparing competent cells to be used in [[Transformation via Electroporation|electroporation transformation]]. | ||
== Materials Needed == | == Materials Needed == | ||
| + | -LB Broth | ||
| + | |||
| + | -28 C incubator | ||
| + | |||
| + | -37 C incubator | ||
| + | |||
| + | -centrifuge | ||
| + | |||
| + | -microfuge tubes | ||
| + | |||
| + | -ice cold 10% glycerol | ||
| + | |||
| + | -micropipette | ||
| + | |||
| + | -cells to be transformed | ||
== Protocol == | == Protocol == | ||
| + | -Inoculate 5 mL of LB broth with bacterial cells to be transformed and grow overnight at 28 C with shaking | ||
| + | |||
| + | -Transfer 2 mL to 200 microliters of LB broth in a 1 liter flask and grow at 37 C with shaking until the optical density is 0.3-0.5 | ||
| + | |||
| + | -Centrifuge the cells at 7,000 rpm for 10 minutes and discard the supernatant | ||
| + | |||
| + | -Resuspend the cells in 35-40 mL of ice cold 10% glycerol and centrifuge at 7,000 rpm for 10 minutes, discard the supernatant | ||
| + | |||
| + | -Resuspend the cells in 18-20 mL of ice cold 10% glycerol and centrifuge at 7,000 rpm for 10 minutes, discard the supernatant | ||
| + | |||
| + | -Resuspend the cells in 350-400 microliters of ice cold 10% glycerol | ||
| + | |||
| + | -Aliquot the cells into small tubes of about 40 microliters each | ||
| + | |||
| + | -The cells can be used right away or frozen in liquid nitrogen and stored for later use | ||
== Notes == | == Notes == | ||
Latest revision as of 15:07, 20 July 2015
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Contents
Purpose
This protocol is used for preparing competent cells to be used in electroporation transformation.
Materials Needed
-LB Broth
-28 C incubator
-37 C incubator
-centrifuge
-microfuge tubes
-ice cold 10% glycerol
-micropipette
-cells to be transformed
Protocol
-Inoculate 5 mL of LB broth with bacterial cells to be transformed and grow overnight at 28 C with shaking
-Transfer 2 mL to 200 microliters of LB broth in a 1 liter flask and grow at 37 C with shaking until the optical density is 0.3-0.5
-Centrifuge the cells at 7,000 rpm for 10 minutes and discard the supernatant
-Resuspend the cells in 35-40 mL of ice cold 10% glycerol and centrifuge at 7,000 rpm for 10 minutes, discard the supernatant
-Resuspend the cells in 18-20 mL of ice cold 10% glycerol and centrifuge at 7,000 rpm for 10 minutes, discard the supernatant
-Resuspend the cells in 350-400 microliters of ice cold 10% glycerol
-Aliquot the cells into small tubes of about 40 microliters each
-The cells can be used right away or frozen in liquid nitrogen and stored for later use
Notes
Papers where this or a similar method has been used
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Related Ontology Terms
References
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