IGEM:PennState/2006/Transformation

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(<font color="green">Transformation</font color>)
Current revision (14:44, 22 May 2008) (view source)
 
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='''Transformation'''=
='''Transformation'''=
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''Time: 2.25 hr''
 
===<font color="blue">Media/Reagents</font color>===
===<font color="blue">Media/Reagents</font color>===
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===<font color="red">Pre</font>===  
===<font color="red">Pre</font>===  
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*Deactivate the ligase by heating at 65 degrees for 20 minutes.
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*Add plasmid DNA to alloquats from electrocompetent cell prep
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*Chill ligated DNA on ice
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*Transfer DNA and Cells into electroporator cuvettes
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*Thaw competent cells ((found in eppendorfs in box on second-shelf down of -80ºC freezer) on ice for ~5 min.
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===<font color="green">Transformation</font color>===
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===<font color="green">Electroporation</font color>===
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#Add 3 μL ligation mixture to cells in bottom of tube
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#Shock Cells, monitor discharge time
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#Incubate on ice for 30 min
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#Add SOC to cuvettes (~400)   
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#Heat shock in 42ºC water bath for 1 min (warm SOC media at same time).  
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#Transfer to culture tubes, shake at 37 for 1 hour (depending on antibiotic marker)
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#Incubate on ice for 2 min
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#Plate ~200uL of SOC/Cells onto agar plate
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#Add 500 μL warm SOC media
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#Shake in 37ºC room for 1 hr
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#Spread 200 μL on LB/antibiotic plates using rotator and glass spreading wand. 
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#Incubate plates in 37ºC room overnight.
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<sup>1</sup>To make SOC, add 20 μL 500 mM filter-sterilized glucose to 500 uL SOB (20 mM final glucose concentration)
<sup>1</sup>To make SOC, add 20 μL 500 mM filter-sterilized glucose to 500 uL SOB (20 mM final glucose concentration)
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[http://openwetware.org/wiki/Penn_State_University_2006:Cloning Cloning]
 
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[http://openwetware.org/wiki/IGEM:PennState Main]
 

Current revision

Contents

Transformation

Media/Reagents

  • Competent cells
  • SOC1
  • Antibiotic LB plates

Pre

  • Add plasmid DNA to alloquats from electrocompetent cell prep
  • Transfer DNA and Cells into electroporator cuvettes

Electroporation

  1. Shock Cells, monitor discharge time
  2. Add SOC to cuvettes (~400)
  3. Transfer to culture tubes, shake at 37 for 1 hour (depending on antibiotic marker)
  4. Plate ~200uL of SOC/Cells onto agar plate

1To make SOC, add 20 μL 500 mM filter-sterilized glucose to 500 uL SOB (20 mM final glucose concentration)

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