Team:Newcastle/Transformation of E. coli

From 2010.igem.org

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(Protocol)
 
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==Materials required==
==Materials required==
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* Appropriate ''E. coli'' strains (200 µl)
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* ''E. coli'' DH5α (200 µl)
* Appropriate vector DNA
* Appropriate vector DNA
* Heat block
* Heat block
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==Protocol==
==Protocol==
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# Thaw a 200 µl aliquot of the desired strain of ''E. coli'' and add the transforming DNA (10 ng of vector DNA in 10 µl).  
+
# Thaw a 200 µl aliquot of ''E. coli'' DH4α and add the transforming DNA (10 ng of vector DNA in 10 µl).  
# Incubate on ice for 30 minutes.
# Incubate on ice for 30 minutes.
# Heat-shock the cells for 120 seconds at 42°C, and place on ice again for 3-4 minutes.
# Heat-shock the cells for 120 seconds at 42°C, and place on ice again for 3-4 minutes.
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# Add 1 ml of LB broth and incubate the cells at 37°C for 1-15 hr in a water bath with gentle shaking.
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# Add 1 ml of LB broth and incubate the cells at 37°C for 1-1.5 hr in a water bath with gentle shaking.
# Plate 200 µl of transformed ''E. coli'' onto 1.5% agar plate containing the appropriate selection markers.
# Plate 200 µl of transformed ''E. coli'' onto 1.5% agar plate containing the appropriate selection markers.
# Incubate the plates overnight at 37°C.
# Incubate the plates overnight at 37°C.

Latest revision as of 14:37, 26 October 2010

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Transformation of E. coli

Materials required

  • E. coli DH5α (200 µl)
  • Appropriate vector DNA
  • Heat block
  • Bucket of ice
  • Pipettes
  • Eppendorf tubes
  • 1.5% agar plate containing appropriate antibiotics

Protocol

  1. Thaw a 200 µl aliquot of E. coli DH4α and add the transforming DNA (10 ng of vector DNA in 10 µl).
  2. Incubate on ice for 30 minutes.
  3. Heat-shock the cells for 120 seconds at 42°C, and place on ice again for 3-4 minutes.
  4. Add 1 ml of LB broth and incubate the cells at 37°C for 1-1.5 hr in a water bath with gentle shaking.
  5. Plate 200 µl of transformed E. coli onto 1.5% agar plate containing the appropriate selection markers.
  6. Incubate the plates overnight at 37°C.


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