Team:UNC Chapel Hill/Procedures

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Compilation of all lab procedures

Contents

Procedure to culture cells

  1. Use culture tubes - rounded bottom, 2 level clamp lid. While incubating, remember to close the lid loosely to allow bacteria to get oxygen.
  2. Add 3mL of carb LB to the culture tube.
  3. Take a clean pipette tip greater than the 3mL level in the tube, touching only the very top of it.
  4. Carefully scoop up a single colony from the agar dish with the pipette tip and drop it into the culture tube. (Once again, make sure the part that you touched does not go into the LB. Use a fairly large pipette tip.)
  5. Label the side of the tube and put into the shaking incubator at 37 degrees C overnight.


Altered Procedure from June 4th

  1. Used LB (Luria Broth: 10g Trypton, 5g yeast extract, 10g NaCl, 1L water) instead of SOC as the growth medium.
  2. Only used 3 Microcentrifuge Tubes with CC cells.


Procedure to create a frozen stock

  1. Add 750µL of bacteria and 250µL of 60% glycerol into a centrifuge tube.
  2. Shake it well to mix.
  3. Label the tube with colored tape.
  4. Put in into the -80 degree C freezer iGem box (2nd row from top, first drawer from left)

Mini-Prep (Protocol pamphlet is available in the lab as well)

  1. Add 1.5µL of bacteria into a small 1.5µL centrifuge tube and label.
  2. Spin in the centrifuge on the highest speed (14,000 rpm) for 1 min.
  3. Add 400µL of lysis.
  4. Put on vortexer until homogenous (~30 sec)
  5. Let sit for 3 min
  6. Spin again for 1 min at 14,000 rpm.
  7. Add 400µL of wash buffer
  8. Spin again for 1 min
  9. Take bottom off and pour out the liquid.
  10. Centrifuge again
  11. Warm up a bottle of elution buffer in microwave for 10 sec
  12. Add 50µL of elution buffer to the column
  13. Spin again
  14. What is left is the DNA. Put into box in -20 degree C freezer.

Optical Density Procedure (0.5µg to 1.0µg of DNA)

  1. In the program, press 'Nucleic Acid'
  2. Put 2µL of water on the bulb on the metal plates and carefully close the jaws
  3. Click 'ok' and 'blank' to calibrate
  4. Wipe off the water and add 2µL of DNA in the same procedure
  5. Click 'Measure'
  6. Record the value
  7. Clean off the bulb and fold a kimwipe between the two jaws before closing

Micro Gel (0.7% gel)

  1. Measure out 0.6g of agarose and pour into a bottle
  2. Add 60mL of TA buffer to the bottle
  3. Microwave the bottle with the lid slightly open until the liquid boils.
  4. Take out the bottle and swirl the solution.
  5. Repeat steps 3 and 4 until all of the agarose is dissolved.
  6. Cool for 2 mins
  7. Add (1µL per 100mL) ethidium bromide
  8. Remove the comb from the plastic holder and pour in a little of the agarose solution and tilt the holder to seal all joints
  9. Put in the comb and pour the rest of the solution in.
  10. Let sit for ~20 mins until solid. (You can blow on the gel and check for lack of ripples to test)
  11. Cut out a piece of parafilm to use for the DNA and dye mixing
  12. Mix 1µL of ladder in 5µL of water and 1µL of loading dye.
  13. Use the above procedure if using a positive control
  14. Mix 1µL of dye with 10µL of DNA for each sample
  15. Inject the mixture vertically and directly into the wells. Make sure the mixtures do not overlap into the next wells
  16. Connect the wires and set the voltage to 120-140V and wait 15-20 mins for the dye to separate into light blue and dark blue colors.
  17. Take out the walls and carry the holder to the -80 freezer room with the UV box
  18. Take out the gel and put it inside the UV box
  19. Set the knob to 'Trans UV'
  20. Open the Kodak MI program on the computer
  21. Click 'Capture GL 200' and then capture.
  22. Export the image to save.
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