Team:BioscienceDragons AZ/Notebook/Materials and Methods

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== '''Procedures outside of iGEM Protocols''' ==
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====='''Agarose gel:'''=====
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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)
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'''Making Agarose gel:'''
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<ul>
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<li>1.5% Agarose gel 100ml(for smaller plates)</li>
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<li>.015g/ml x 100ml TAE = 1.5g Agarose</li></ul>
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'''Ethdium Bromide gel:'''Just add 5-10microliters of ethidium bromide to gel after it cools
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====='''Competent cells:'''=====
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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.
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'''Procedure:'''
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<ul>
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<li> 1.Place overnight culture on ice for 20min </li>
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<li> 2.Centrifuge for 5min (then decant the supernatant)</li>
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<li> 3. Add in 5ml of .1M of CaCl2 </li>
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<li> 4. Place on ice for 30min </li>
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<li> 5. Centrifuge for 5min (then decant supernatant)</li>
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<li> 6. Store at 0- (-4) degrees celsius (one day lifespan)</li>
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</ul>
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'''Transformation procedure:'''
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<ul>
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<li> 1. Add 50microliters of competent cells to 2 mini centrifuge tubes </li>
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<li> 2. Add 1 microliter of DNA to one tube and none to the other (control) </li>
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<li> 3. Place tubes on ice for 30mins </li>
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<li> 4. Heat shock for 45seconds in 42° celsius water bath </li>
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<li> 5. Transfer to ice for 2mins </li>
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<li> 6. Add 500microliters of LB-broth per tube </li>
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<li> 7. Place at 37° celsius rotating incubator for overnight. </li>
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<li> 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. </li>
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</ul>

Revision as of 03:17, 22 June 2013


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.
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