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Team:BIOTEC Dresden/ProtocolsAHL assay
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(New page: {{Biotec_Dresden/Header}} <html> <body> <div id="content_prim"> <div id="materials"> <h2>Materials</h2> <ul> <li>Escherichia coli DH5 alpha chemical competent cells</li> <li>1mL SOC (room ...)
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(New page: {{Biotec_Dresden/Header}} <html> <body> <div id="content_prim"> <div id="materials"> <h2>Materials</h2> <ul> <li>Escherichia coli DH5 alpha chemical competent cells</li> <li>1mL SOC (room ...)
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Revision as of 00:39, 28 October 2010
Materials
- Escherichia coli DH5 alpha chemical competent cells
- 1mL SOC (room temperature) for each reaction
- Ice
- Plasmid DNA
- Heating block
- 1.5ml microfuge tube
- LB-agar plate with corresponding antibiotic
Procedure
- Chill DNA samples and tubes on ice.
- Place LB-agar plates in 37°C incubator to warm.
- Remove chemical competent cells from -80°C freezer and thaw on ice. Alternatively, freshly prepared chemical competent cells may be used immediately.
- Dial a P2 pipetman to either 1 or 2μL depending on the salt content of your DNA sample. Use 2μL for samples that have been purified in some way.
- Dial a P200 pipetman to 50μL or whatever volume of chemical competent cells you want to use; usually 20-50μL .
- Pipet 1-2μL of DNA sample and add to chemical competent cells. Swirl tip around gently in cells to mix DNA and cells. Do not pipet up and down.
- Place the mix back on ice for 30 mins.
- Pulse the cells with a heat shock by placing the microfuge tubes in a heating block for 45 seconds at 42°C.
- Place sample on ice for 2 mins and then add SOC medium. This step should be done as quickly as possible to prevent cells from dying off.
- Chill sample on ice for 2 mins to permit the cells to recover.
- Transfer eppendorf tube to 37°C incubator and shake to promote aeration. Incubate for 1 hr to permit expression of antibiotic resistance gene.
- Plate transformation onto prewarmed LB-agar plate supplemented with appropriate antibiotic.
Reference
adapted from http://openwetware.org/wiki/Knight:Electroporation
