IGEM:UNAM-Genomics-Mexico/2009/Notebook/iGEM 2011 UNAM-Genomics Mexico/2011/05/24

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Transformation of the PBBRMCS-5 and PSB1T3 Ligation into DH5α

The ligation and the controls are transformed into E. coli DH5α bacteria. We expect that the ligation transformation will yield red bacteria with a gentamicin resistance.

Transformation method

We have four tubes with 100 μL each one with E. coli DH5α competent cells.

The Tubes are numbered in the following way:

  1. 100 μL of E. coli DH5α competent cells with 10 μL of undigested PBBRMCS-5 plasmid.
  2. 100 μL of E. coli DH5α competent cells with 10 μL of digested PBBRMCS-5 plasmid with XBA I and Hind III phosphated.
  3. 100 μL of E. coli DH5α competent cells with 10 μL of digested PBBRMCS-5 plasmid with XBA I and Hind III dephosphated.
  4. 100 μL of E. coli DH5α competent cells with 10 μL of digested PBBRMCS-5 plasmid with XBA I and Hind III dephosphated, ligated with the PCR amplification of PSB1T3.
  • Incubate in ice for 30 minutes.
  • Thermic Shock for 1 minute at 42ºC.
  • Leave the tubes in ice for 5 minutes.
  • Add 1 mL of liquid LB.
  • Incubate the tubes for one hour at 37ºC and agitate at 250 rpm.
  • Centrifugate during 4 minutes at 4000 rpm
  • Plate in petri dishes with solid LB and gentimicin (for every mL of LB 1μL of gentamicin)200 μL per dish.



Leave over night at 37ºC.


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