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From 2013hs.igem.org

Contents 1 Procedures outside of iGEM Protocols 1.1 Agarose gel: 1.2 Competent cells: 1.3 Transformation procedure:

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.