Purdue/25 June 2008

From 2008.igem.org

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Second Attempt at Transformations

As the first transformation attempt failed, we're trying again (with the same parts--I732017 and J22106).

Obtained a new set of Chemically Compotent Cells from Larisa. Cells are considered Max Efficiency.

Used a slightly different protocol, at Jessamine's suggestion:

  • DNA Prep:
    • Punched out spots, soaked in 5 uL of warm TE for 2 min.
    • Vortexed 5s, spun down 30s
    • Added 5uL more TE
    • Vortexed and spun down again
    • Got as much liquid as possible (~5uL) from the eppendorfs, transferred to 1.5mL Eppendorfs
    • Put on ice for less than 5 minutes
  • Transformation
    • Added DNA to 25uL of just-thawed cells (Better than last time when they sat out for a while)
    • Let sit on ice (no mixing) for 30 min.
    • Heat shocked at 42C for 65s, put on ice
    • Added 1mL LB, incubated at 37C for 1 hour
    • Spin down, eliminate all but 200uL of LB
      • Plate 100uL of DNA/LB onto Amp agar plate, let grow o/n at 37C
      • Put 100uL of DNA/LB into 3mL of LB, shake at 37C o/n (let grow without antibiotics to help recovery)

UV Vis Spectroscopy Data from Plasmid

Utilized UV Vis in Room 234 to get an absorbance reading from DNA plasmid to determine the amount of DNA on a single punch from the part kit.

Analyzation revealed approximately 112 ug/mL, which translates to 1 ug per spot (or punch). The second transformation above used one spot of DNA.

Edited by Craig Barcus and Janie Stine