IGEM:UNAM-Genomics-Mexico/2009/Notebook/iGEM 2011 UNAM-Genomics Mexico/2011/05/31

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PBBRMCS-5 DNA Plasmid extraction and Digestion

PBBRMCS-5 was extracted using alcaline lysis and 15μL Plasmid DNA digested with XBA I and Hind III

PBBRMCS-5 Plasmid DNA extracttion

We used Miguel Angel Ramirez's Method without the steps 11, and 13 through 20.

Gel Electrophoresis

The gel was made with 100 mL of TAE 1X and 1 gr. of Agarose, to have a gel with 1% agarose.

  • 1st Lane -> 5 μL of Ladder (Fermentas)
  • 2nd Lane -> 5 μL PBBRMCS-5 DNA Plasmid, 2 μL loading DNA dye. (1)
  • 3rd Lane -> 5 μL PBBRMCS-5 DNA Plasmid, 2 μL loading DNA dye. (2)
  • 4th Lane -> 5 μL PBBRMCS-5 DNA Plasmid, 2 μL loading DNA dye. (3)
  • 5th Lane -> 5 μL PBBRMCS-5 DNA Plasmid, 2 μL loading DNA dye. (4)
  • 6th Lane -> 2 μL of Loading DNA Dye.
  • 7th Lane -> 5 μL of Ladder (Fermentas)

The gel ran for 50 minutes at 110 volts

PBBRMCS-5 Plasmid Digestion

  • 2.5 μL of H20
  • 2.5 μL of NEBuffer 2 10X
  • 1.25 μL of XBA I
  • 1.25 μL of Hind III
  • 2.5 μL of BSA
  • 15 μL of PBBRMCS-5 Plasmid DNA

For a total reaction of 25 μL.

Leave all night at 37ºC in agitation.