"

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.