Team:Gaston Day School/Experiment

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Experiments and Protocol

Labels

For MCF tubes:

  1. P: Plasmid Prep
  2. D: Digest
  3. T: Transformation
  4. L: Ligation

Transformation

  1. x = the number of transformations you are doing
  2. Place the SOC medium (from the fridge) in the incubator
  3. Also place X plates in the incubator (With the appropriate antibiotic resistance, Chl for complete, Amp for parts)
  4. Make an ice-water bath
  5. Obtain X MCF tubes and a float
  6. Add 50 ul of competent cells
  7. Add 2ul of the DNA
  8. Place it in the ice-water bath for 2 mins
  9. While waiting, turn on the sink's hot water, obtain a beaker, add hot water, and using a thermometer, adjust water to 42o C (using the hot plate if needed)
  10. Place tubes in the incubator for 30 seconds
  11. Another 2 mins on ice
  12. Add 1 ml of warm SOC medium
  13. Place each tube in the incubator (37o C) for 1 hour
  14. Plating

Pick Colonies

  1. Obtain the green sticks, empty LB tubes, and the plates from the incubator
  2. Label the tubes with corresponding plates
  3. Spot the colonies (Choose more than one if available)
  4. Use the sticks and gently obtain the colonies
  5. Swirl around in the LB solution and dispose the stick into the tip trash
  6. Put the tubes into the shaking incubator overnight

Plasmid Prep Procedure

  1. TENS procedure (make fresh each time)-To 4.5 ml TE solution add 250 ul 10% SDS and 250 ul 2M NaOH
  2. Transfer 1.5ml bacterial culture to a labelled MCF tube
  3. Centrifuge for 30 seconds to pellet bacteria
  4. Pour off most of the growth medium. Leave approximately 100ul in the tube
  5. Resuspend the pellet by shaking, vortexing, or tapping the tube vigorously. Make sure the bacteria are completely resuspended and no clumps remain before continuing.
  6. Add 100ul TENS to the tube
  7. Mix well by inversion for 2 minutes. Do not shake the tube. The solution will get viscous
  8. Add 150ul sodium acetate and mix well
  9. Centrifuge for 3 minutes
  10. Transfer the supernatant (liquid) to a clean, labeled MCF tube
  11. Add 1ml 75% Ethanol to the tube and mix well by inversion. You may see DNA as faint white strands in the liquid. The tube should be nearly full of liquid. Sharpie brand pens have an ink that is soluble in Ethanol. Make sure your labels do not get removed accidentally.
  12. Centrifuge 5 minutes to pellet the DNA. Place the tubes in the centrifuge with the hinges out to make finding the pellet easier
  13. Pour off the 95% Ethanol
  14. Place the tubes upside-down and allow them to dry completely
  15. Resuspend the pellet in 25 uL TE
  16. Use 10 uL of the DNA in a restriction digest or 1 uL for PCR

Digest

  1. obtain the plasmid prep tubes (in incubator), new MCF tubes, enzymes in the freezer
  2. For each new tube, prepare the master mix(MM):
    • water 15ul
    • buffer 2ul
    • Upstream enzyme 0.5ul
    • Downstream enzyme 0.5ul
  3. Each tube needs 18ul of the MM, in order to get enough MM into each tube. If you have x digest, do x+1 MM. (Ex. If I need to do a digest for one tube, I will prepare 30ul of MM, but still put 18ul into the tube)
  4. Label the new tubes with corresponding tubes and change the "P" into a "D"
  5. Add 3ul DNA and 15ul MM into each tube
  6. Obtain a tube float and put all the tubes into the dry incubator for at least an hour.

 

Email: iGEM@gastonday.org

Gaston Day School

2001 Gaston Day School Rd

Gastonia 28056