Team:UNC Chapel Hill/Procedures

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Compilation of all lab procedures

Contents

Procedure to make plates

Purpose: Create plates where bacterial colonies can grow after transformation. Selects for bacteria that generate antibiotic resistance (e.g. carb resistance). Notes: Use only bacterial plates, not tissue plates

  1. Measure out 4.5g of agar
  2. Add to 500mL of LB
  3. Mix/swirl
  4. Autoclave
    1. Leave the lid loose
    2. Close the autoclave tightly
    3. Hit '#4' for liquid cycle
    4. Wait for ~20 mins
  5. Swirl again
  6. Add the appropriate amount of antibiotic to make a 1x solution (500mL -> 500µL)
  7. Wait for it to cool until you can hold it for ~10 seconds
  8. Pour into plates and pop any bubbles with the Bunsen burner. Bubbles that go to the edge do not need to be popped.

Procedure to transform/plate bacteria

Purpose: Allows the bacteria to take up the iGem part or another plasmid of interest and then be plated for purpose of selecting bacteria that took up the plasmids.

Beginning

Notes: Keep all microcentrifuge and Eppendorf tubes on ice unless indicated otherwise!

  1. Find the iGem part of interest. If the part solution hasn't been made (aka not in blue box), then puncture the appropriate well and inject 15 µL of dH20. Let sit for a couple of minutes to homogenize. The liquid should turn to a solid color due to an indicator in the well.
  2. Add all of the parts mixture (dH20 and part) to the PCR tubes and place them into the iGem blue freezer box (in the -20 C freezer). This is where we are keeping stock of the iGem parts. This is a very important step. Input their location into the [http://uncbme.com/igem/index.php?page=parts parts database].
  3. Label Eppendorf Tubes and sit them in ice for each transformation. This is where the bacteria will be going followed by individual parts. It's important for them to stay cold, so that when the bacteria is transferred, the bacteria will not be damaged.
  4. Find microcentrifuge tubes for bacteria of interest from the -80 C freezer, usually DH5α-E Chemically competent bacteria [http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Transformation/Chemically-Competent.html] or maybe the Electrocompetent bacteria.
  5. Let the bacteria thaw in the ice until a slurry. Place 25 µL of bacteria into all the appropriate Eppendorf tubes (most likely all of them).
  6. Add 1 µL of iGem part liquid into the appropriate tubes. Feel free to also have one tube for a positive control (e.g. pBluescript). This will allow you to make sure that the bacteria is good.

Chemically-Competent Transformation

Follow these steps if using Chemically-Competent bacteria. (If you're not sure, Chemically-competent is usually the default)

  1. Let the CC cells sit for 10 minutes. Afterward, heat shock the tubes at 42 degrees Celsius for 30 seconds each. Then add 500 µL of LB (Luria Broth: 10g Trypton, 5g yeast extract, 10g NaCl, 1L water) growth medium WITHOUT antibiotic to each tube and put them back onto ice.

Electroporation Transformation

Follow these steps if using Electroporated Bacteria

  1. Place bacteria into individual electroporation cuvettes. Electroporate tubes at 50 µF, 150 ohms, 1.5 kVolts by pulsing twice. Immediately add 500 µL of LB or SOC growth medium to each after shocking and place back onto ice.

Afterwards

  1. Incubate all the tubes in a shaker for about 1 hour at 37 degrees Celsius. Use a piece of tape to append tubes to the bottom of the shaker.
  2. Be sure to put the bacteria stock back. You may want to slow freeze to ensure that the bacteria do not die out. This is accomplished by taking the bacteria stock and putting a styrofoam box with dry ice until it become frozen enough.
  3. Plate 50 µL of bacteria on a corresponding agar dish with the appropriate resistance (usually carbenicillin). Spread around with curved glass pipets.
  4. Incubate overnight in the oven. Check for colonies the next day to see if iGem plasmid was taken up. If there are colonies, then that means the transformation worked; otherwise, you will have to repeat the procedure again.

Procedure to culture bacteria

Purpose: After getting colonies from the previous procedure, you'll want to culture those colonies into a sufficient stock so that you can create a frozen stock, test for plasmid uptake, etc.

  1. Use culture tubes - rounded bottom, 2 level clamp lid.
  2. Label the side of the tube
  3. Add 3mL of LB + antibiotic to the culture tube using electronic pipette.
  4. Take a clean pipette tip greater than the 3mL level in the tube, touching only the very top of it.
  5. Carefully scoop up a single colony from the agar dish with a p1000 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.)
  6. While incubating, remember to close the lid LOOSELY to allow bacteria to get oxygen. You will know the cap is loose if the cap moves between two points freely. Careful not to tip the tube.
  7. Put into the shaking incubator at 37 degrees C overnight.
  8. Parafilm the agar dishes and put in fridge. This is so that you have a backup in case the culturing does not go as well as expected. Feel free to throw out these dishes when you get successful cultures.

Procedure to create a frozen stock

Purpose: Create frozen stock of a culture of transformed bacteria. This can be stuck in a -80 C freezer and stay good for years.

  1. Label a twist cap centrifuge tube with colored tape and a black sharpie. It's generally better to write out the label on the piece of tape, cut with the razor, and then adhere it to the outside of the tube.
  2. Add 750µL of bacteria and 250µL of 60% glycerol
  3. Shake it well to mix. No need for Vortex; this can be done by hand.
  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)

Purpose: Allows you to isolate the DNA, which can then be used for Gel Electrophoresis.

  1. Add 1.5mL of bacteria into a small 1.5mL centrifuge tube and label.
  2. Spin in the centrifuge on the highest speed (14,000 rpm) for 1 min.
  3. Pour out the liquid carefully, making sure the seed remains at the bottom.
  4. Add 400µL of lysis.
  5. Put on vortexer until homogenous (~30 sec)
  6. Let sit for 3 min
  7. Pour contents into a spin colum tube.
  8. Spin again for 1 min at 14,000 rpm.
  9. Empty bottom part of column.
  10. Add 400µL of wash buffer
  11. Spin again for 1 min
  12. Empty bottom part of column.
  13. Add 400µL of wash buffer
  14. Spin again for 1 min
  15. Take the top off and put into an eppendorf tube.
  16. Warm up a bottle of elution buffer in microwave for 10 sec
  17. Add 50µL of elution buffer to the column
  18. Spin again
  19. What is left in the eppendorf tube is the DNA. Put into box in -20 degree C freezer.

QIAGEN Mini-Prep

  1. Resuspend pelleted bacterial cells in 250µL Buffer P1 and transfer to a micro-centrifuge tube.
    1. Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.
  2. Add 250µL Buffer P2 and gently invert the tube 4-6 times to mix. Mix gently by inverting the tube. Do not vortex, as this would result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and clear. Do not allow the lysis reaction to proceed for more than 5 minutes.
  3. Add 350µL Buffer N3 and invert the tube immediately but gently 4-6 times. To avoid localized precipitaion, mix the solution gently but throughly, immediately after addition of Buffer N3. The solution should become cloudy.
  4. Centrifuge for 10 minutes at 13,000 rpm (~17,900 x g). A compact white pellet will form.
  5. Apply the supernatant from step 4 to the quiprep spin colum by decanting or pipetting.
  6. Centrifuge for 30-60 seconds. Discard the flow-through.
  7. Optional: Wash the quiaprep spin column by adding 0.5mL Buffer PB and centrifuge for 30-60 seconds. Discard the flow-through.
    1. Necessary when using endA+ strains, such as JM series, HB101, or any wild-type which have high levels of nucleus activity.
  8. Wash quiaprep spin column by adding 0.75mL Buffer PE and centrifuging for 30-60 seconds.
  9. Discard the flow-through and centrifuge for additional minute to remove residual wash buffer.
  10. Place the quiaprep column in a clean 1.5mL micro-centrifuge tube. To elude DNA. add 50uL Buffer EB (10mM Tris-Cl, pH 8.5) or water to the center of each quiaprep spin column, let stand for 1 min, and centrifuge for 1 min.

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

Purpose: Allows you to measure the density of the DNA in the MiniPrep solution.

  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

Gel Electrophoresis (Micro 0.7% gel)

Purpose: Gel Electrophoresis is a classic procedure to compare the sizes of fragments of DNA. If you know that your fragment should be say 3 kB, then gel electrophoresis will allow you to verify that.

  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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