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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