Team:BioscienceDragons AZ/Notebook/Materials and Methods

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Contents

Procedures outside of iGEM Protocols

Agarose gel:

Agarose gels are used to separate strands of DNA depending on their size. Agarose is a carbohydrate taken from algae that when combined with TAE buffer and made into a gel makes little microscopic pores for DNA to pass through. The agarose gel is placed in an electrophoresis machine and a current of electricity is run through it to pell the DNA from the negative to the positive end.(since DNA is negatively charged)

Making Agarose gel:

  • 1.5% Agarose gel 100ml(for smaller plates)
  • .015g/ml x 100ml TAE = 1.5g Agarose

Ethdium Bromide gel:Just add 5-10microliters of ethidium bromide to gel after it cools

Competent cells:

Use CaCl2 breaks down the cell wall to increase permeability, and neutralize the cells charge. then heat shocking is used to make the pores of the cell open wide.

Procedure:

  • 1.Place overnight culture on ice for 20min
  • 2.Centrifuge for 5min (then decant the supernatant)
  • 3. Add in 5ml of .1M of CaCl2
  • 4. Place on ice for 30min
  • 5. Centrifuge for 5min (then decant supernatant)
  • 6. Store at 0- (-4) degrees celsius (one day lifespan)


Transformation procedure:
  • 1. Add 50microliters of competent cells to 2 mini centrifuge tubes
  • 2. Add 1 microliter of DNA to one tube and none to the other (control)
  • 3. Place tubes on ice for 30mins
  • 4. Heat shock for 45seconds in 42° celsius water bath
  • 5. Transfer to ice for 2mins
  • 6. Add 500microliters of LB-broth per tube
  • 7. Place at 37° celsius rotating incubator for overnight.
  • 8. Plate 50 microliters of each tube on separate agar plates, the control on a regular plate and the other on a plate with its respective antibiotic.