Team:Jefferson VA SciCOS/Records

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Growing Bacteria on Agar Plates

1. Wash hands; wear lab coat; wipe down the table with a cleaning solution. (70% ethanol)

2. Obtain a bunsen burner and light it.

3. Obtain clean ready-made agar plates containing ampicillin resistance and/or chloramphenicol resistance from the refrigerator. To distinguish the ampicillin plates, there is a blue stripe on the side of the plate. For the chloramphenicol plates, there is a strip of paper on the plate labeling it as “chlor”.

4. Use a permanent marker to label the bottom of the agar plate with the date, type of bacteria, and iGEM.

5. Obtain an already sterilized inoculating loop from the iGEM distribution kit or from Dr. Burnett.

6. Obtain a few cells from the agar stab and gently streak the plate in a zig-zag fashion along one quadrant of the plate. Obtain more cells and drag the end of the first zig-zag along an adjoining quadrant of the plate to make another zig-zag pattern. Repeat this procedure to make two more zig-zags, to cover all four quadrants of the plate.

7. Close the lid, and use parafilm to close the edges of the plate.

8. Place plates upside down in a 37 degrees Celsius incubator and incubate for 15-20 hours. Transfer the plates to a fridge when they are finished incubating.

9. Check on the plates to make sure enough colonies have grown!

Making a Liquid Culture

1. Wash hands; wear lab coat; wipe down the table with a cleaning solution (70% ethanol)

2. Obtain a bunsen burner and heat it.

3. Obtain ready-made liquid LB from the refrigerator and crushed ampicillin from Dr. Burnett.

4. Add 5 ml of the LB add 5 µl of ampicillin to a large tube. This results in a concentration of 100 µg/ml for the broth.

5. Using a sterile inoculating loop, select a single colony from the KGF, FGF, and oxygen promoter plates.

6. Drop the inoculating loop into the LB broth + ampicillin mixture and swirl.

7. Loosely cover the culture with parafilm.

8. Incubate the bacterial culture for 37 degrees Celsius for 12-18 hours in a shaking incubator.

9. After incubation, check for growth, which is characterized by a cloudy haze in the media.

Miniprep Note: We followed the 3A Assembly protocol exactly for the miniprep procedure. For convenience, we have included the procedure here.

1. Spin the cell culture in a centrifuge to pellet the cells, empty the supernatant (media) into a waste collection container.

2. Resuspend pelleted bacterial cells in 250 µl Buffer P1 (kept at 4 degrees Celsius) and transfer to a microcentrifuge tube. No cell clumps should be visible after resuspension of the pellet.

3. Add 250 µl Buffer P2 and gently invert the tube 4-6 times to mix. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.

4. Add 350 µl Buffer N3 and invert the tube immediately and gently 4-6 times. To avoid localized precipitation, mix the solution gently but thoroughly, immediately after addition of Buffer N3. The solution should become cloudy.

5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A white pellet will form.

6. Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.

7. Centrifuge for 30-60 s. Discard the flow-through.

8. Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 60 s. Discard the flow-through.

9. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 60 s.

10. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.

11. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl Buffer EB or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

12. Label the microcentrifuge tube lightly with “KGF” or “oxygen promoter”.

Restriction Digest

1. Keep all enzymes and buffers on ice

2. Thaw 10x buffer at room temperature. Flick and spin the liquid to collect it at the bottom of the tube.

3. Obtain two eppendorf tubes and label one of them “KGF” and the other “OX Prom”

4. To two fresh eppendorf tubes, add 10 µl of DNA to each. Add distilled water to the tubes for a total volume of 16 µl in each tube.

5. Add 2 µl of 10x buffer to each tube.

6. To the OX Prom tube, add 1 µl of EcoR1 and 1 µl of Spe1. To the KGF tube, add 1 µl of EcoR1 and 1 µl of Xba1.

7. The total volume in the tubes should be 20 µl. Mix the contents of the tubes by pipetting up and down slowly. Spin the samples briefly to collect all of the mixture at the bottom of the tube.

8. Using a thermal cycler, incubate the restriction digests at 37 degrees Celsius for 30 minutes, then at 80 degrees Celsius for 20 minutes.

9. Keep the restriction digest tubes in the refrigerator until use.

Ligation

1. Keep all enzymes and buffers on ice.

2. Thaw T4 DNA Ligase buffer at room temperature. Flick and spin the liquid to collect it at the bottom of the tube.

3. Obtain one small tube (specific to the thermal cycler) and label it KGF + Pro.

4. To the tube, add 5 µl of distilled water, 2 µl of T4 DNA Ligase Buffer, 1 µl of T4 DNA Ligase, and 6 µl of each of the digested parts.

5. The total volume in the tubes should be 20 µl. Mix the contents of the tubes by pipetting up and down slowly. Spin the samples briefly to collect all of the mixture at the bottom of the tube.

6. Using a thermal cycler, incubate the restriction digests at 16 degrees Celsius for 30 minutes, then at 80 degrees Celsius for 20 minutes.

7. Keep the ligation tube on ice until use.

Transformation Link to [http://openwetware.org/wiki/BioBuilding:_Synthetic_Biology_for_Students:_Lab_4 What a Colorful World Lab protocol]

Part 1: Preparing Competent Cells

1. Label two small eppendorf tubes “Ex” and “Control”

2. Pipet 200 µl of CaCl2 into each eppendorf tube and place the tubes on ice.

3. Use a sterile inoculating loop to scrape up a patch of cells from the NEB-beta culture grown earlier and swirl the cells in the cold CaCl2.

4. Resuspend the cells by vortexing them briefly.

5. Keep the competent cells on ice while preparing for transformation.

Part 2: The Transformation

1. Make one 5 µl aliquot of the ligated part.

2. Flick the tube with the competent NEB-beta strain and pipet 75 µl of the bacteria into the tube with the 5 µl aliquot. Pipet 75 µl of bacteria into a clean eppendorf tube that will serve as a control. Label the tube with the 5 µl aliquot as “Ex” and the other as “Control”.

3. Let the tubes sit on ice for 5 minutes. Use a timer to count the time.

4. While the DNA and cells are incubating, label the bottoms of the 2 LB+amp petri dishes. Label one of the plates as “Ex, iGEM, date” and the other as “Control, iGEM, date”

5. Heat shock each of the samples by placing the tubes at 42 degrees Celsius for 90 seconds exactly.

6. At the end of the 90 seconds, move the tubes to a rack at room temperature.

7. Pipet 250 µl of the experimental transformation mix onto the experimental plate. Pipet 250 µl of the control transformation mix onto the control. Spread the mix across the entire plate using a sterile inoculating loop.

8. Use parafilm to seal the edges of the plates.

9. Incubate the petri dishes with the agar side up at 37 degrees Celsius overnight.