IGEM:UNAM-Genomics-Mexico/2009/Notebook/iGEM 2011 UNAM-Genomics Mexico/2011/06/02

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Digestion Control

We ligated the product of the digestion to make sure the plasmid PBBRMCS-5 was indeed digested with both enzymes (XBA I and Hind III). Then we transformed the plasmid with E. coli DH5α.

We expect that the transformation will not yield many E. coli colonies (there is not a perfect digestion).

Ligation

  • H2O -> 5.5 μL
  • Buffer 10X with DNTPs -> 1 μL
  • DNA (Plasmid) -> 2.5 μL
  • T4 ligase -> 1 μL
Ligation Control
  • H2O -> 8 μL
  • Buffer 10X with DNTPs -> 1 μL
  • T4 ligase -> 1 μL

For two total reactions of 10 μL.

Leave the reaction for two hours at 25ºC

Transformation into E. coli DH5α

  • We have 3 tubes with 100 μL of competent E. coli DH5α cells:
     * Tube 1 -> Transformation Control (no Plasmid DNA added)
     * Tube 2 -> 4 μL of ligated plasmid DNA added
     * Tube 3 -> 4 μL of the ligation control added (no plasmid DNA added)
  1. Leave in ice for 20 minutes
  2. Heat shock for 1 minute at 42ºC
  3. Leave in ice for 5 minutes
  4. Add 1 mL of liquid LB to each of the three tubes
  5. Incubate for one hour at 37ºC
  6. Plate in petri dishes with solid LB and gentamicin
  7. Leave overnight at 37ºC and check in the morning for colonies




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