Team:UNC Chapel Hill/Procedures

From 2009.igem.org

Revision as of 19:17, 8 July 2009

Compilation of all lab procedures

Procedure to make carb plates

Notes: Used only for 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 carb 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

Procedure to plate cells

Notes:

• Keep Microcentrifuge tubes on ice unless indicated otherwise!

1. Find the iGem part of interest. It has a promoter, RBSs, terminators, and a RFP (Red Fluorescent Protein) Puncture the appropriate well (O6 on the 2nd Set) and injected 15 µL of dH20. Let sit for a couple of minutes to homogenize. The liquid should turn red due to an indicator in the well.
2. Label six Microcentrifuge Tubes numbers 1-6 and sit them in ice. It's important for them to stay cold, so that when the bacteria is transferred, the bacteria will not be damaged.
3. Find microcentrifuge tubes for bacteria of interest from the freezer, in this case: DH5α-E Chemically competent bacteria [http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Transformation/Chemically-Competent.html] and Electocompetent bacteria
4. Let the bacteria thaw in the ice until a slurry. Place 25 µL of bacteria into the appropriate tube (CC in 1 through 3; EC in 4 through 6)
5. Add 1 µL of iGem part liquid into the appropriate tubes (1 and 4). Likewise for the pBluescript (2 and 5)
6. 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 carb antibiotic to each tube and put back onto ice.
7. Place 4-6 into individual electroporation cuvettes. Electroporate tubes 4-6 at 50 µF, 150 ohms, 1.5 kVolts by pulsing twice. Immediately add 500 µL of SOC growth medium to each after shocking and place back onto ice.
8. Incubate all the tubes in a shaker for about 1 hour at 37 degrees Celsius.
9. Plate 50 µL of bacteria on a corresponding agar dish with ampicillin or carbenicillin. Spread around with curved glass pipets.
10. Incubate overnight in the oven. Check the colonies the next day to see if iGem plasmid was taken up.

Procedure to culture cells

1. Use culture tubes - rounded bottom, 2 level clamp lid.
2. Label the side of the tube
3. Add 3mL of carb LB to the culture tube.
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 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.)
6. While incubating, remember to close the lid LOOSELY to allow bacteria to get oxygen.
7. Put into the shaking incubator at 37 degrees C overnight.

Procedure to create a frozen stock

1. Label a twist cap centrifuge tube with colored tape and a black sharpie
2. Add 750µL of bacteria and 250µL of 60% glycerol
3. Shake it well to mix.
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 1.5mL 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.

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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