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:
- 100 μL of E. coli DH5α competent cells with 10 μL of undigested PBBRMCS-5 plasmid.
- 100 μL of E. coli DH5α competent cells with 10 μL of digested PBBRMCS-5 plasmid with XBA I and Hind III phosphated.
- 100 μL of E. coli DH5α competent cells with 10 μL of digested PBBRMCS-5 plasmid with XBA I and Hind III dephosphated.
- 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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