IGEM:Harvard/2007/week 0/Protocol

• Day 1: Transformation protocol (6/11/07)

1. Thaw the 6 tubes of cells on ice and mix gently to ensure that the cells are evenly suspended.
   1. OMPA1 in PET
   2. OMPA1 + his in PET
   3. OMPA1 + strep2 in PET
   4. OMPA2 in PET
   5. OMPA2 + his in PET
   6. OMPA2 + strep in PET
2. Add 1 µl of the DNA solution directly to the cells. Stir gently to mix.
3. Place the tubes on ice for a little more than 5 minutes
4. Heat the tubes for about 45 seconds in a 42°C water bath; do not shake
5. Place on ice for 2 minutes
6. Add 250 µl of room temperature SOC Medium to each tube
7. Shake at 37°C (300 rpm) for more than 30 minutes.
8. Light the bunsen burner
9. Using two sets of petri dishes containing LB agar w/ Kanamycin (1 w/ 5 µl of cells and one w/ 50 µl of cells):
   1. 5 µl: pipette 5 µl of cells with 45 µl of SOC onto the agar mix
   2. 50 µl: pipette 50 µl of cells onto the agar mix.
10. For each plate, we use sterile technique to spread the agar:
    1. Take the metal spreader and soak the bottom in ethanol, making sure to rinse exposed parts of the handle with distilled water.
    2. Very briefly put the ethanol soaked parts of the spreader under the flame.
    3. Touch the agar to make sure the spreader isn't too hot (will singe if too hot), and spread the cells around the plate.
11. Afterwards, turn the plates upside down and incubate at 37°C

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• Day 2: Growing Bacteria in Liquid Medium (6/12/07)

1. Take out the 6 grown colonies.
2. Get 6 100 mL culture tubes.
3. Light the bunsen burner.
4. In each tube, use the pipet gun to add 2 mL of KB, and add 2 µl of Kanamycin (50 mg/mL)1000x
   1. Make sure to lightly graze the tip of the KB with the top unscrewed a little for additional sterilization
5. Briefly place the tongs under the flame to sterilize, and use them to pick up a sterile toothpick.
6. Use the toothpick to gather a single colony of cells, and place the toothpick and the colony into the solution.
7. Repeat this sterile technique with the other 6.
8. Shake at 37°C (300 rpm) for more than 30 minutes.
9. Incubate overnight

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• Day 3: DNA Plasmid Preps

1. 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.
2. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.
   1. Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.
   2. 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.
3. Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times.
   1. 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.
   2. 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.
4. Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times.
   1. 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.
   2. 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.
5. Centrifuge for 10 min.
   1. A compact white pellet will form.
6. Apply the supernatants that resulted from step 4 to the QIAprep spin column by decanting or pipetting.
7. Centrifuge for a minute. Discard the flow-through.
8. Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for a minute. Discard the flow-through.
   1. This step is necessary to remove trace nuclease activity when using endA+ strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content. Host strains such as XL-1 Blue and DH5α™ do not require this additional wash step.
9. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for a minute. Discard flow-through.
10. Centrifuge for an additional 1 min to remove residual wash buffer.
    1. Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions.
11. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

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• Day 4:

Digestion of OmpA1 protocol (6/14/07)

1. Add 20ul OmpA1 in pET29b+
2. Add 3ul 10x NEB buffer 2
3. Add 1ul Nhe1
4. Add 1ul Pst1
5. Add 0.3ul BSA 100x
6. Add 4.7ul H2O
   1. This should amount to 30ul total
7. In a PCR machine:
   1. Incubate for 4 hrs at 37°C
   2. Heat inactivate for 20 min at 65°C
   3. Store overnight at 4°C

Digestion of OmpA2 protocol (6/14/07)

1. Take out 3 PCR tubes (2 for OMPA1 and 1 for OMPA2 because of differences in volume)
2. Add 20ul OmpA2 in pET29b+
3. Add 3ul 10x NEB buffer 2
4. Add 1ul Nhe1
5. Add 1ul Pst1
6. Add 0.3ul BSA 100x
7. Add 4.7ul H2O
   1. This should all amount to 30ul total
8. In a PCR machine:
   1. Incubate for 4 hrs at 37°C
   2. Heat inactivate for 20 min at 65°C
   3. Store overnight at 4°C

Master Mix

• 12ul 10x NEB buffer 2
• 4ul Nhe1
• 4ul Pst1
• 1.2ul BSA 100x
• 18.8ul H2O

• Day 4 (additional): Growing Bacteria in Liquid Medium (in larger quantities)

1. See day 2 protocol, but with 165 mL of LB and 165 µl Kanamycin

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• Day 5: Vector Dephosphorylation Protocol

1. Mixture for OmpA1
   1. 70 ul of OmpA1
   2. 8 ul of Antarctic Phosphatase Reaction Buffer (10x)
   3. 2 ul of Antarctic Phosphatase
   4. Total of 80 ul

1. Mixture for OmpA2
   1. 45 ul of OmpA2
   2. 5.5 ul of Antartic Phosphatase buffer
   3. 2 ul of Antartic Phosphatase
   4. 2.5 ul of water

1. Incubate for 2 hrs at 37°C
2. Heat inactivate (or as required to inactivate the restriction enzyme) for 5 minutes at 65°C.
3. Proceed with ligation.

• Day 5 (additional): DNA Plasmid Prep with Midi Kit

1. Harvest the bacterial cells by centrifugation at 8000 RPM for 15 min at 4°C
2. Resuspend the bacterial pellet in 4 ml of Buffer P1.
3. Add 4 ml of Buffer P2, mix thoroughly by vigorously inverting the sealed tube 4–6 times, and incubate at room temperature (15–25°C) for 15 min.
4. Add 4 ml of chilled Buffer P3, mix immediately and thoroughly by vigorously inverting 4–6 times, and incubate on ice for 15 min.
5. Centrifuge at 17000 RPM for 30 min at 4°C. Remove supernatant containing plasmid DNA promptly.
6. Centrifuge the supernatant again at 17000 RPM for 15 min at 4°C. Remove supernatant containing plasmid DNA promptly.
7. Equilibrate a QIAGEN-tip 100 by applying 4 ml Buffer QBT, and allow the column to empty by gravity flow.
8. Apply the supernatant from step 8 to the QIAGEN-tip and allow it to enter the resin by gravity flow.
9. Wash the QIAGEN-tip with 2 x 10 ml or Buffer QC.
10. Elute DNA with 5 ml Buffer QF.
11. Precipitate DNA by adding 3.5 ml room-temperature isopropanol to the eluted DNA. Mix and centrifuge immediately at 11000 RPM for 30 min at 4°C. Carefully decant the supernatant.
12. Wash DNA pellet with 2 ml of room-temperature 70% ethanol, and centrifuge at 11000 RPM for 10 min. Carefully decant the supernatant without disturbing the pellet.
13. Air-dry the pellet for 5–10 min, and redissolve the DNA in a suitable volume of buffer (e.g., TE buffer, pH 8.0, or 10 mM Tris·Cl, pH 8.5).

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Protocol Recorded by Kevin Shee

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