IGEM:Harvard/2007/week 1

Day 8: Transformation protocol (6/18/07)

1. Thaw the 4 tubes of cells on ice and mix gently to ensure that the cells are evenly suspended. We will be using the following DNA:
   1. OMPA1 + his in PET
   2. OMPA1 + strep2 in PET
   3. OMPA2 + his in PET
   4. 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, use sterile beads to spread the bacteria over the mix.
11. Afterwards, turn the plates upside down and incubate at 37°C

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Day 8: Nucleotide Removal (6/18/07)

1. Add 10 volumes of Buffer PN to 1 volume of random library extensions (single primer) and mix.
2. Place a QIAquick spin column in a provided 2 ml collection tube.
3. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min at 6000 rpm.
4. Discard the flow-through and place QIAquick column back into the same tube.
5. To wash QIAquick column, add 750 µl of Buffer PE and centrifuge for 1 min at 6000 rpm.
6. Discard the flow-through and place the QIAquick column back in the same tube, which should be empty. Centrifuge for an additional 1 min at 13,000 rpm (~17,900 x g).
7. Place the QIAquick column in a clean 1.5 ml microcentrifuge tube.
8. To elute DNA, add 100–200 µl of Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the center of the QIAquick membrane and centrifuge the column for 1 min at 13,000 rpm (~17,900 x g). Alternatively, for increased DNA concentration, add 30–50 µl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge.

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Day 9:Digestion of OmpA1 protocol (6/19/07)

1. Add 15ul OmpA1 in pET29b+
2. Add 3ul 10x NEB buffer2
3. Add 1ul Nhe1
4. Add 1ul Pst1
5. Add 0.3ul BSA 100x
6. Add 9.7ul H20
   1. This should all 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

Day 9:Digestion of OmpA2 protocol (6/19/07)

1. Add 15ul OmpA2 in pET29b+
2. Add 3ul 10x NEB buffer2
3. Add 1ul Nhe1
4. Add 1ul Pst1
5. Add 0.3ul BSA 100x
6. Add 9.7ul H20
   1. This should all 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

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Day 9: Nucleotide Removal 2 (6/19/07)

1. Add 10 volumes of Buffer PN to 1 volume of random library extensions (2 primers) and mix.
2. Place a QIAquick spin column in a provided 2 ml collection tube.
3. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min at 6000 rpm.
4. Discard the flow-through and place QIAquick column back into the same tube.
5. To wash QIAquick column, add 750 µl of Buffer PE and centrifuge for 1 min at 6000 rpm.
6. Discard the flow-through and place the QIAquick column back in the same tube, which should be empty. Centrifuge for an additional 1 min at 13,000 rpm (~17,900 x g).
7. Place the QIAquick column in a clean 1.5 ml microcentrifuge tube.
8. To elute DNA, add 100–200 µl of Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the center of the QIAquick membrane and centrifuge the column for 1 min at 13,000 rpm (~17,900 x g). Alternatively, for increased DNA concentration, add 30–50 µl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge.

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Day 9: Vector Dephosphorylation Protocol Again (6/19/07)

1. Mixture for OmpA1 (2)
   1. 30 ul of OmpA1
   2. 5 ul of Antarctic Phosphatase Reaction Buffer (10x)
   3. 2 ul of Antarctic Phosphatase
   4. 13 ul of H2O
      1. Total of 50 ul

1. Mixture for OmpA2 (2)
   1. 30 ul of OmpA2
   2. 5 ul of Antartic Phosphatase buffer
   3. 2 ul of Antartic Phosphatase
   4. 13 ul of water
      1. Total of 50 ul

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.

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Day 9: Setting up and running gels (6/19/07)

1. For large DNA strands (~5000), you need 1% wt/vol of ultrapure agarose. For small DNA strands (~80), you need 2% wt/vol of ultrapure agarose.
2. Solution: 1xTBE
3. Add 1% wt of ultrapure agarose into TBE solution.
4. Add 5ul EtBr
5. Stir by shaking and then microwave at 30 seconds intervals, stopping to check if it is boiling. Make sure you stop at exact boiling point. Let cool while you set up mold.
6. Level the base, and put in the mold.
7. Put in the comb
8. Pour the gel
   1. Add 5 ul loading dye into each sample (10x dye)
   2. Add 2.5 ul of sample
9. Making sure the proper ends are connected, run gel for ~20 minutes at 150 V.

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Day 10: Gel Extraction (6/20/07)

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 µl).
   1. Lane 2 - OMPA1 - 0.210 g
   2. Lane 3 - OMPA1 - 0.212 g
   3. Lane 4 - OMPA1 - 0.269 g
   4. Lane 5 - OMPA1 - 0.214 g
   5. Lane 6 - OMPA1 - 0.206 g
   6. Lane 7 - OMPA2 - 0.238 g
   7. Lane 8 - OMPA2 - 0.320 g
   8. Lane 9 - OMPA2 - 0.320 g
   9. Lane 10 - OMPA2 - 0.122 g
   10. Lane 11 - OMPA2 - 0.124 g
3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation.
4. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).
5. Add 1 gel volume of isopropanol to the sample and mix.
6. Place a QIAquick spin column in a provided 2 ml collection tube.
7. To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min.
8. Discard flow-through and place QIAquick column back in the same collection tube.
9. Add 0.5 ml of Buffer QG to QIAquick column and centrifuge for 1 min.
10. To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min.
11. Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at ≥10,000 x g (~13,000 rpm).
12. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
13. Elute DNA: Add water (50 ul for most and 30 ul for less concentrated samples - lanes 10 and 11) to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min.

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Day 10: Growing Bacteria in Liquid Medium (6/20/07)

1. Take out the grown colonies.
   1. OMPA1 + his
   2. OMPA1 + strep
   3. OMPA2 + his
   4. OMPA2 + strep
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 10: Ligation Reaction of OmpA1 and OmpA2 with the Random Library (6/20/07)

1. Add 1 ul of 10x T4 DNA Ligase Buffer
2. Add 6.5ul of digested Omp random PCR
3. Add 2ul of digested 1)OmpA1 or 2)OmpA2
4. Add 0.5ul T4 DNA ligase
5. Set up the following reactions:
   1. OmpA1 + Random Library from PCR (B)
   2. OmpA1 + Random Library from Extension (S)
   3. OmpA2 + Random Library from PCR (B)
   4. OmpA2 + Random Library from Extension (S)

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Day 10: Transformation protocol of ligated DNA (6/20/07)

1. Thaw the 4 tubes of cells on ice and mix gently to ensure that the cells are evenly suspended. We will be using the following DNA:
   1. OmpA1 + Random Library from PCR (B)
   2. OmpA1 + Random Library from Extension (S)
   3. OmpA2 + Random Library from PCR (B)
   4. OmpA2 + Random Library from Extension (S)
2. Add 5 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, use sterile beads to spread the bacteria over the mix.
11. Afterwards, turn the plates upside down and incubate at 37°C

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Day 11: Bacterial Innoculation and Induction (6/21/07)

Using 3 sets:

1. Add 5 ul of Kanamycin
2. Add 5 ml of LB
3. Add 100 ul of bacteria:
   1. OmpA1 + his
   2. OmpA1 + strep
   3. OmpA2 + his
   4. OmpA2 + strep
4. Add 5 ul of IPTG
   1. One set add none
   2. One set add immediately
   3. One set add when the culture is at log phase.

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Day 11: Measuring Optical Density to measure cell density (6/21/07)

1. With each different kind of cell (1 or 2 with strep or his), make 6 different cultures:
   1. 0 dilution: 1000 ul of cells w/ 0 ul LB
   2. 2 dilution: 500 ul of cells w/ 500 ul LB
   3. 4 dilution: 250 ul of cells w/ 750 ul LB
   4. 8 dilution: 125 ul of cells w/ 875 ul LB
   5. 16 dilution: 62.5 ul of cells w/ 937.5 ul LB
   6. LB solution: 0 ul of cells w/ 1000 ul LB
2. Using the Genesis UV scanning machine, we will measure the OD of the samples to find log phase:
   1. Pipette culture into the clear tubes, making sure the clear sides are facing the circle.
   2. Insert blank (LB only), 0, 2, 4, 6, 8, and 16 into the machine.
   3. Press measure blank (should go to zero)
   4. Log phase is between 0.2 and 0.8 OD, so watch the absorption value until it hits these values.
      1. See absorbance page for exact values calculated by the team.
3. When the bacteria enters log phase, add IPTG to the sample that needs IPTG added.
4. Then we're ready to induce.

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Day 11: Tranformation of cells that have had more growth time (6/21/07)

• See Day 10 Protocol.
• See Plates for results of colonies.

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Day 11: Running Protein Gel of Induced Bacteria Outer Membrane Proteins (6/21/07)

1. With the induced bacteria, spin down 100 ul of each sample.
2. Pipette out the liquid (mostly LB), leaving the pellet at the bottom of the tube.
3. Resuspend with 100 ul TBS
4. In separate tubes, add:
   1. 4 ul cell/TBS mix
   2. 5 ul dye
   3. 16 ul TBS
5. Boil mixture for 2 minutes
6. Prepare gel:
   1. Remove comb and tape from gel
   2. rinse wells with distilled water.
   3. To help see the gel, mark a dot at the botton of the well on the long plate side
   4. Pour buffer to cover the gel in the middle compatment
   5. Pour buffer to halfway for the outer compartments
7. Load gel lane 1 with 10 ul ladder
8. Load gel lanes 2-9 with 20 ul of the different samples.
9. Repeat gel process for the next set of mixtures.
10. once the dye has run approximately 70 % of the gel's length, you can stop the process
11. rinse in distilled water while continuously and gently tipping the container -- allow 10 minutes for each wash to rid of the sds
12. then stain with Gelcode Blue Stain Reagent -- add enough to cover the gel submerged in the water

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Day 12: Miniprep of ligated colonies (6/22/07)

1. Plate the following ligated colonies:
   1. OmpA2 with Random Library (alpha and beta)
   2. OmpA1 with Random Library + ext.
   3. OmpA2 with Random Library + ext.
2. Take and spin the tubes with 1 mL in each for 1 minute at high speed.
3. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.
4. Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times.
5. Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times.
6. Centrifuge for 10 min.
7. Apply the supernatants that resulted from step 4 to the QIAprep spin column by decanting or pipetting.
8. Centrifuge for a minute. Discard the flow-through.
9. Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for a minute. Discard the flow-through.
10. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for a minute. Discard flow-through.
11. Centrifuge for an additional 1 min to remove residual wash buffer.
12. 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.
13. Finally, measure the concetrations of the DNA using the NanoDrop® ND-1000 Protocol, and store them in the freezer.

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Day 12: Ligation Reaction for Overnight (6/22/07)

1. Add 1 ul of 10x T4 DNA Ligase Buffer
2. Add 6.5ul of digested Omp random PCR
3. Add 2ul of digested 1)OmpA1 or 2)OmpA2
4. Add 0.5ul T4 DNA ligase
5. Set up the following reactions:
   1. OmpA1 + Random Library from PCR (B)
   2. OmpA1 + Random Library from Extension (S)
   3. OmpA2 + Random Library from PCR (B)
   4. OmpA2 + Random Library from Extension (S)

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Day 12: Growing Bacteria in Liquid Medium (6/22/07)

1. Take out two well-grown colonies
   1. OMPA1 + library (extension)
   2. OMPA1 + library (PCR)
2. Get 10 100 mL culture tubes
   1. 5 for each
3. Light the bunsen burner.
4. In each tube, use the pipet gun to add 2 mL of LB, and add 2 µl of Kanamycin (50 mg/mL)1000x
   1. Make sure to lightly graze the tip of the LB 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 9.
8. Shake at 37°C (300 rpm) for more than 30 minutes.
9. Incubate overnight

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Protocol Recorded by Kevin Shee