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Team:BioscienceDragons AZ/Notebook/Materials and Methods
From 2013hs.igem.org
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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. | 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. | ||
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<li> 1. Add 50microliters of competent cells to 2 mini centrifuge tubes </li> | <li> 1. Add 50microliters of competent cells to 2 mini centrifuge tubes </li> | ||
Revision as of 03:17, 22 June 2013
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.
