IGEM:Harvard/2007/Protocols
iGEM Protocol for 2007
Day 1: Transformation protocol (6/11/07)
- Thaw the 6 tubes of cells on ice and mix gently to ensure that the cells are evenly suspended.
- OMPA1 in PET
- OMPA1 + his in PET
- OMPA1 + strep2 in PET
- OMPA2 in PET
- OMPA2 + his in PET
- OMPA2 + strep in PET
- Add 1 µl of the DNA solution directly to the cells. Stir gently to mix.
- Place the tubes on ice for a little more than 5 minutes
- Heat the tubes for about 45 seconds in a 42°C water bath; do not shake
- Place on ice for 2 minutes
- Add 250 µl of room temperature SOC Medium to each tube
- Shake at 37°C (300 rpm) for more than 30 minutes.
- Light the bunsen burner
- Using two sets of petri dishes containing LB agar w/ Kanamycin (1 w/ 5 µl of cells and one w/ 50 µl of cells):
- 5 µl: pipette 5 µl of cells with 45 µl of SOC onto the agar mix
- 50 µl: pipette 50 µl of cells onto the agar mix.
- For each plate, we use sterile technique to spread the agar:
- Take the metal spreader and soak the bottom in ethanol, making sure to rinse exposed parts of the handle with distilled water.
- Very briefly put the ethanol soaked parts of the spreader under the flame.
- Touch the agar to make sure the spreader isn't too hot (will singe if too hot), and spread the cells around the plate.
- Afterwards, turn the plates upside down and incubate at 37°C
Day 2: Growing Bacteria in Liquid Medium (6/12/07)
- Take out the 6 grown colonies.
- Get 6 100 mL culture tubes.
- In each tube, use the pipet gun to add 2 mL of KB, and add 2 µl of Kanamycin (50 mg/mL)1000x
- Light the bunsen burner.
- Briefly place the tongs under the flame to sterilize, and use them to pick up a sterile toothpick.
- Use the toothpick to gather a single colony of cells, and place the toothpick and the colony into the solution.
- Repeat this sterile technique with the other 6.
- Shake at 37°C (300 rpm) for more than 30 minutes.
- Incubate overnight
Day 3: DNA Plasmid Preps
- Take out the 6 tubes of grown bacteria in liquid medium and spin two tubes with 1 mL in each for 1 minute at high speed.
- Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge
tube.
- Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible
after resuspension of the pellet.
- If LyseBlue reagent has been added to Buffer P1, vigorously shake the buffer bottle
to ensure LyseBlue particles are completely dissolved. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
- Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times.
- Mix gently by inverting the tube. 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.
- If LyseBlue has been added to Buffer P1 the cell suspension will turn blue after addition
of Buffer P2. Mixing should result in a homogeneously colored suspension. If the suspension contains localized colorless regions or if brownish cell clumps are still visible, continue mixing the solution until a homogeneously colored suspension is achieved.
- Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube
4–6 times.
- To avoid localized precipitation, mix the solution thoroughly, immediately after
addition of Buffer N3. Large culture volumes (e.g. ≥5 ml) may require inverting up to 10 times. The solution should become cloudy.
- If LyseBlue reagent has been used, the suspension should be mixed until all trace
of blue has gone and the suspension is colorless. A homogeneous colorless suspension indicates that the SDS has been effectively precipitated.
Protocol Recorded by Kevin Shee
