Team:Kyoto/Protocols

Protocols

Index

• Construction
  • Solubilization of Antibiotics
  • Media
  • Transformation
  • Miniprep
  • Restriction Digestion
  • PCR
  • Electrophoresis
  • Gel Extraction
  • Ligation
  • Dephosphorylation
  • Sequence
  • Ethanol Precipitation
• Measurement
  • Promoter Activity (from 24 August to 15 September)
  • Promoter Activity (from 16 September)
  • GFP Fluorescence (from 24 August to 17 October)
  • Promoter Activity and GFP Fluorescence (from 18 October)
  • Measurement of the actitvity of inducible lytic system

Construction

Solubilization of Antibiotics

1. Mix the following (Final concentration is 50mg/mL).
   • Ampicillin:

     Ampicillin

     1.0g

     MilliQ

     20mL

   • Kanamycin:

     Kanamycin

     0.5g

     MilliQ

     10mL

2. Dispense 1.1mL of the solution into 1.5mL tubes.
3. Store in the -20℃ freezer.

Media

LB Media

1. Wash a graduated cylinder with MilliQ.
2. Add the following to about 180mL of MilliQ in the graduated cylinder:

   Bacto-yeast extract

   1.0g (0.5w/v%)

   Tryptone

   2.0g (1.0w/v%)

   NaCl

   2.0g (1.0w/v%)

   1N NaOH

   200µL

3. Seal the graduated cylinder by a Parafilm.
4. Dissolve the mixture by inverting the graduated cylinder.
5. Adjust volume to 200mL by adding more MilliQ.
6. Add the media solution to a 200mL Erlenmeyer flask.
7. (To prepare solid media, add 2.4g (1.2w/v%) of agar to the flask.)
8. Wrap the tops of the flasks with aluminum foil.
9. Place a small piece of auto clave tape on one of the flasks.
10. Autoclave the media on liquids.
11. Store at room temperature.
12. To pour plates, melt the solid media in the microwave, then place flask in water bath to bring media to 50℃.
13. Add 200µL of antibiotic

    Ampicillin

    100µg/mL

    Kanamycin

    50µg/mL

14. (Add 0.5 - 1.0w/v% Glucose for protein expression.)

Supplemented M9 Media

1. Wash a graduated cylinder with MilliQ.
2. Add the following to about 180mL of MilliQ in the graduated cylinder:

   Na2HPO4

   1.2g (0.6w/v%)

   KH2PO4

   0.6g (0.3w/v%)

   NaCl

   0.1g (0.05w/v%)

   NH4Cl

   0.2g (0.1w/v%)

3. Seal the graduated cylinder by a Parafilm.
4. Dissolve the mixture by inverting the graduated cylinder.
5. Adjust volume to 200mL by adding more MilliQ.
6. Add the medium solution to a 200mL Erlenmeyer flask.
7. Wrap the tops of the flasks with aluminum foil.
8. Place a small piece of auto clave tape on one of the flasks.
9. Autoclave the media on a liquids.
10. Cool at room temperature.
11. When it become as cool as you can hold it,Add the following to it:

    MgSO4

    1.0mM（final cocentration）

    CaCl2

    0.1mM（final concentration）

    thiamine hydrochloride

    0.001%（final concentration）

    casamino acids

    0.2w/v%

12. Add 200µL of antibiotic:

    Ampicillin

    100µg/mL

    Kanamycin

    50µg/mL

13. Add 0.4w/v% Glucose.

Transformation

1. Unfreeze conpitent cells on ice.
2. Dry a plate by letting the plate upside down and partly open in incubator.
3. Add 1µL DNA solution and 20µL compitent cells to 1.5mL tube, let stand for 30min on ice. If few colony is observed, increase the amount of the compitent cells or DNA, but make the amount of DNA not to get over that of the compitent cells.
4. Heatshock for 60s at 42℃.
5. Let stand for 2min on ice.
6. Culture for 1h in preculture medium (LB or SOC medium), and plate by using spreader. Do not heat spreader too much because e.coli will dead for heat.

Miniprep

1. Use QIAprep Spin Miniprep Kit Cat. No. 27104 by QIAGEN
2. Pick a single colony from a freshly streaked selective plate and inoculate a culture of about 3mL LB medium containing the appropriate selective antibiotic.
3. Incubate at 170rpm for 8h at 37℃ with vigorous shaking.
4. Transfer a half of the culture to a tube.
5. Harvest the bacterial cells by centrifugation at 14,000g for 1min at 4℃. Remove the medium by decanting.
6. Transfer the half of the culture to same tube and harvest as same. Remove the medium by pippetting.
7. Resuspend pelleted bacterial cells in 250µL Buffer P1 and mix thoroughly by pippeting.
8. Add 250µL Buffer P2 and mix thoroughly by inverting the tube gently 4-6 times.
9. Add 350µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
10. Centrifuge for 10min at 14,000g at 4℃.
11. Apply the supernatants from step 10 to the QIAprep spin column by pipetting.
12. Centrifuge for 10s in a table-top microcentrifuge. Discard the flow-through.
13. Wash the QIAprep spin column by adding 0.5mL Buffer PB and centrifuging for 10s in a table-top microcentrifuge. Discard the flow-through.
14. Wash QIAprep spin column by adding 0.65mL Buffer PeE and centrifuging fo 10s in a table-top microcentrifuge.
15. Discard the flow-through, and centrifuge for and additional 1min to remove residual wash buffer.
16. Place the QIAprep column in a clean tube. To elute DNA, add 50µL water to the center of each QIAprep spin column, let stand for 1min, and centrifuge for 1min.
17. Discard the QIAprep spin column.
18. Measure the concentration of DNA by using eppendorf BioPhotometer plus.
19. Restriction Digestion.
20. Agarose Gel Electrophoresis for Confirmation.

Restriction Digestion

1. Use EcoRI, XbaI, SpeI, PstI, DpnI（NEB）
2. Mix the following.

   Sample

   5µL

   10xBuffer

   1µL

   Restriction Enzyme

   0.1µL

   MilliQ

   3.9µL

3. Let stand for 2h at 37℃

PCR

Standard PCR

1. Dilute template DNA. If the concentration of DNA is 2-100ng/µL, transfer 1µL to a clean tube and add 99µL MilliQ.
2. Dilute Primer. If the concentration of Primer is XµM, dilute primer X timers and transfer 1µL to a clean tube and add 99µL MilliQ.
3. Mix the following.
   1. For use of KOD plus ver2:

      25mM MgSO4

      3µL

      2mM dNTPs

      5µL

      10xBuffer for KOD plus ver.2

      5µL

      Template DNA (5ng/µL)

      5µL

      Primer Forward (10µM)

      1.5µL

      Primer Reverse (10µM)

      1.5µL

      KOD plus ver.2

      1µL

      MilliQ

      28µL

      Total

      50µL

   2. For use of KOD FX:

      2mM dNTPs

      10µL

      2xBuffer for KOD FX

      25µL

      Template DNA

      5µL

      Primer Forward (10µM)

      1.5µL

      Primer Reverse (10µM)

      1.5µL

      KOD FX

      1µL

      MilliQ

      6µL

      Total

      50µL

4. (If amplification is not succeeded, try at 4.5 or 6.0µL 25mM MgSO4.)
5. Let stand for 2min at 94℃.
6. 25-40 cycles for 10s at 98℃, for 30s at Tm-5℃, and for 1min (1min for 1kb) at 68℃ (Tm is temparature at which primer will dissolve).
7. At 15℃ forever.
8. Agarose Gel Electrophoresis for confirmation.

Screening PCR

1. Mix the following (Do on PCR Bench).

   10x PCR buffer (TAKARA)

   40µL

   2.5mM dNTP

   8µL

   Primer-1 (10pmol/µL)

   8µL

   Primer-2 (10pmol/µL)

   8µL

   Ex Taq HS (TAKARA)

   1.6µL

   MilliQ 

   334µL (to total 400µL)

2. Dispense 25µL to 15 tubes.
3. Pick a single colony and transfer it to each tubes.
4. Suspend the colony.
5. Let stand for 10min at 90℃.
6. 35 cycles for 30s at 94℃, for 30s 55℃, and for 1min at 72℃.
7. Let stand for 4min at 72℃.
8. Add 5mL Loading Buffer to the tubes.
9. Agalose Gel Electrophoresis for confirmation.
10. Negative Control: Use nothing.
11. Positive Control: Use a colony that will yield a product with this primers.

Mutagenesis (Point mutation, Deletion, Insertion)

1. Mix the following.

   10xBuffer

   5µL

   2mM dNTP

   5µL

   Primer Forward (10µM)

   1.5µL

   Primer Reverse (10µM)

   1.5µL

   Template Plasmid (50ng/µL)

   1µL

   KOD plus ver.2

   1µL

   MilliQ

   35µL

   Total

   50µL

2. Prepare control: instead of KOD plus ver.2, add 1µL MilliQ.
3. Let stand for 2min at 94℃.
4. X cycles (1 cycle for 1kb) for 10s at 98℃ and for Ymin (1min for 1kb) at 68℃.
5. Hold at 4℃.
6. Take 25µL of the solutions into fresh tubes.
7. Add 1µL DpnI (10U/µL).
8. Let stand for 1h at 37℃.
9. Agarose gel electrophoresis, using 5µL of the solution for confirmation.
10. Mix the following.

    Sample

    2µL

    Ligation high

    5µL

    T4 Polynucleotide Kinase (5U/µL)

    1µL

    MilliQ

    7µL

    Total

    15µL

11. Let stand for 1h at 16℃.
12. Transformation, using 10µL of the solution.

PCR Purification

1. Use QIAquick PCR Purification Kit Cat. No. 28104 by QIAGEN
2. Add BufferPB about 5 times as much as the product of PCR.
3. Apply the solution to the column.
4. Centrifuge for 1min at 1300rpm. Discard the flow-through. If the amount of the sample is much, repeat this step by same column.
5. Add 750µL BufferPE and let stand for 2-3min. If the solution overflows, we decrease the amount of BufferPE.
6. Centrifuge for 1min and discard the through.
7. Centrifuge for additional 1min to remove residual buffer.
8. Place the column in a clean tube.
9. Add 10µL BufferEB or MilliQ to the center of each column, let stand for 1min, and repeat this step.
10. Centrifuge for 1min at 13000rpm.
11. Discard the column.

Electrophoresis

1. Prepare 200mL of a 1.0% agarose solution:
2. Measure 2.0g agarose into a beaker.
3. Add 200mL 1xTAE buffer.
4. Wrap the top of the beaker with plastic wrap.
5. Punch a hole through the wrap with a pipette tip (To let out steam).
6. Dissolve the agarose by heating in microwave and swirling without boiling.
7. Allow the agarose to cool.
8. Pour the agarose solution into a gel tray on a gel maker.
9. If there is air bubbles, pushing them with a pipette tip.
10. Place comb in the maker.
11. Cover the maker with a plastic wrap.
12. Let stand for about 45min.
13. Remove the comb carefully.
14. Store in the Tupperware in the refrigerator.
15. Place the tray in electrophoresis chamber.
16. Cover the tray with 1xTAE buffer.
17. To prepare samples for electrophoresi, add 1µL of 6x Loading Buffer for every 5µL of DNA solution and mix well.
18. Load 6µL of the DNA solution per well.
19. Electrophorese at 100V for about 30min until Loading Buffer have migrated approximately three-quaters of the gel.
20. Stain the gel in 0.5µg/mL ethidium bromide for 20-30min.
21. Rinse the gel with MilliQ.
22. Place a plastic wrap on the transilluminator in the cabinet of Printgraph.
23. Place the gel on the transilluminator.
24. Turn on the transilluminator and confirm the position of the gel.
25. Shoot the picture.
26. Turn off the transilluminator.
27. Dispose of the gel.

Gel Extraction

1. Use QIAquick Gel Extraction Kit Cat. No. 28704 by QIAGEN
2. Transfer cutted gel to a tube.
3. Add BufferQG about 3 times as much as the volume of the gel.
4. Stand the gel for 10min at 50℃ to Dissolve. If hard to dissolve, sometimes vortex.
5. Confirm the color of the solution. If the color is orange or purple, add about 10µL 3M sodium acetate to yellow.
6. Add isopropanol as much as the gel and mix.
7. Apply the solution to the column.
8. Centrifuge for 1min at 1300rpm. Discard the flow-through. If the amount of the sample is much, repeat this step by same column.
9. Add 500µL BufferQG and centrifuge for 1min. Discard the through.
10. Add 750µL BufferPE and let stand for 2-3min. If the solution overflows, we decrease the amount of BufferPE.
11. Centrifuge for 1min and Discard the through.
12. Centrifuge for additional 1min to remove residual buffer.
13. Place the column in a clean tube.
14. Add 10µL BufferEB or MilliQ to the center of each column, let stand for 1min, and repeat this step.
15. Centrifuge for 1min at 13000rpm.
16. Discard the column.

Ligation

1. Make 2µL of Mixture (the vector and the insert at 1 : 5-10) and Control (only the vector).
2. Add 5µL Ligation High, 1µL T4 Kinase, and 7µL MilliQ to create a solution.
3. Incubate at 16℃ for 30 min. If the colonies of E.coli transformed with the Control,

Dephosphorylation

1. Use Bacterial Alkaline Phosphatase (BAP, Takara)
2. Set the heating block at 65℃
3. Mix the following:

   MilliQ

   24µL

   10x AP Buffer

   5µL

   DNA sample

   20µL

   BAP

   1µL

   total

   50µL

   • Not need to purify the DNA sample.
4. Incubate the solution at 65℃ for 30min.
5. Add 50µL MilliQ to the solution (Total is 100µL).
6. Add 100µL mixed lipid of Phenol and Chloroform (at 1:1).
7. Vortex the solution sufficiently.
8. Centrifuge for 5min at 15,000rpm at room temparature and transfer the supernatant to a tube.
9. Repeat the previous step a few times.
10. Add 100µL Chloroform.
11. Vortex the solution.
12. Centrifuge for 5min at 15,000rpm at room temparature and transfer the supernatant to a tube.
13. Add 10µL 3M Sodium Acetate and 250µL 100% cold Etanol and mix them.
14. Let stand for 15min on the ice.
15. Centrifuge for 15min at 15,000rpm, 4℃ and discard the supernatant.
16. Add 500µL 70% Etanol.
17. Centrifuge for 5min at 15,000rpm, 4℃ and discard the supernatant.
18. Dry it.
19. Add appropriate quantiles (about 20µL) of TE Buffer to dissolve the DNA.
20. Stored at -20℃.

Sequence

1. Use Big Dye Terminator 3.1（ABI）
2. Mix the following

   5xBuffer

   2µL

   Primer (3.2µM)

   1µL

   Template Plasmid 

   200ng

   Big Dye Terminator 3.1

   0.5µL

   MilliQ

   up to 10µL

3. Let stand for 1min at 96℃.
4. 35 cycles for 5s at 98℃, for 5s 50℃, and for 2.5min at 68℃.
5. Add 25µL 100% ethanol and 1µL NAOAC

Ethanol Precipitation

1. Use Ethachinmate (NIPPON GENE、312-01791).
2. Add 3.3 µL of 3M Sodium Acetate (attached with Ethachinmate) into 100µL of DNA solution.
3. Add 1µL of Ethachinmate.
4. Vortex.
5. Add ethanol, 200-250µL.
6. Vortex.
7. Centrifuge at 12000xg for 5min.
8. Precipitation.

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Measurement

Promoter Activity (08/24-09/15)

1. Pour 5mL the supplemented M9 medium to each test tube.
2. Add 100µL 0.025M IPTG aq to each test tube and mix well.
3. Pick out a colony on the plate of E.coli and put into the test tube.
4. Incubate the culture at 37℃ for 16h.
5. Measure OD600 of the overnight culture.
6. Pour (25/OD600) µL the culture to 1.5mL tube.
7. Centrifuge the tube at 14,000rpm and at 4℃ for 2min.
8. Discard the supernatant.
9. Pour 1mL the supplemented M9 medium to the tube and mix well to dissolve pellet.
10. Do procedures 7-10 again.
11. Pour 25mL the supplemented M9 medium to test tube.
12. Add proper amount of 0.025M IPTG aq to the test tube to make the medium including IPTG concentration you wish.
13. Add 1mL the medium including IPTG to the pellet in the tube from the test tube and dissolve it.
14. Add the all culture in the tube to 24mL the medium in the test tube and make the culture including IPTG concentration you wish. OD600 of this culture is 1.0×10-3.
15. Divide 25mL the culture to 5 test tubes by 5mL to measure OD600 and to collect samples of the measurement of GFP fluorescence at 5 different times.
16. Incubate these test tubes at 37℃.
17. Measure OD600 of the culture after 4h, 8h, 12h, 16h and 20h.
18. Pour 1mL of the culture to 1.5mL tube at the times.
19. Centrifuge the 1.5mL tube at 14,000rpm and at 4℃ for 2min.
20. Discard the supernatant.
21. Freeze the pellets and store it in a fridge as samples for measurement of GFP fluorescence.
22. Measure GFP fluorescence.

Promoter Activity (09/16-10/17)

1. Pour 5mL the supplemented M9 medium to each falcon tube.
2. Add proper amount of 0.025M IPTG aq to each test tube and mix well to make the medium including IPTG concentration which you hope.
3. Pick out a colony on the plate of E.coli and put into three falcon tubes.
4. Incubate the culture at 37℃ for 16h.
5. Measure OD600 of the overnight culture.
6. Pour 5mL the supplemented M9 medium to each new falcon tube. Pre-warm these tubes at 37℃.
7. Pour 50µL the overnight culture to 5mL the fresh medium.
8. Incubate the diluted culture at 37℃.
9. Measure OD600 of the culture after 2h, 2.5h, 3h, 3.5h and 4h.
10. Pour 1mL of the culture to 1.5mL tube after 3h and 3.5h.
11. Centrifuge the 1.5mL tube at 14,000rpm and at 4℃ for 2min.
12. Discard the supernatant.
13. Freeze the pellets and store it in a fridge as samples for measurement of GFP fluorescence.
14. Measure GFP fluorescence.

GFP Fluorescence (from 24 August to 17 October)

1. Add 75µL the cell lysis reagent to the pellet in the 1.5mL tube and mix well and dissolve it.
2. Centrifuge the tube at 15,000rpm and at 4℃ for 1min.
3. Take out 50µL supernatant and add to each well of a plate.
4. Add 50µL the cell lysis reagent to a well to correct the back.
5. Set the plate in the plate reader, Wallac 1420 Multilabel Counter, and measure GFP fluorescence of the samples (Ex/Em = 485/535nm, 1sec).

Promoter Activity and GFP Fluorescence (from 18 October)

1. Pour 5mL the supplemented M9 medium to each falcon tube.
2. Add proper amount of 0.025M IPTG aq to each test tube and mix well to make the medium including IPTG concentration which you hope.
3. Pick out a colony on the plate of E.coli and put into three falcon tubes.
4. Incubate the culture at 37℃ for 16h.
5. Measure OD600 of the overnight culture.
6. Pour 5mL the supplemented M9 medium to each new falcon tube. Pre-warm these tubes at 37℃.
7. Pour 50µL the overnight culture to 5mL the fresh medium.
8. Incubate the diluted culture at 37℃.
9. Measure OD600 of the culture after 2h, 2.5h, 3h, 3.5h and 4h.
10. Pour 300µL of the culture to 1.5mL tube after 3h and 3.5h and cool it on ice.
11. Add 200µL the culture to each well of a plate.
12. Add 200µL the supplemented M9 medium to a well to correct tha back.
13. Set the plate in the plate reader, Wallac 1420 Multilabel Counter, and measure GFP fluorescence of the samples (Ex/Em = 485/535nm, 1sec).

Quantitative measurement of lytic activity of λ lysis cassette

1. Pour 3mL of supplemented M9 medium to a Falcon tube and add certain amount of IPTG.
2. Pick out three colony on the plate of E.coli and put into the Falcon tube.
3. Grow it at 30 degreees for 20h.
4. Measure OD550 when time of incubation is 16h,18h, and 20h.
5. Confirm that the values of OD550 of cultures incubated 16h, 18h, 20h doesn't change.We define it as a parameter of lytic activity, LAV（Lytic avtivity value）.

Measurement of lytic activity with time

1. Pour 3mL of supplemented M9 medium to a Falcon tube.
2. Pick out a colony on the plate of E.coli and put into the Falcon tube.
3. Grow it at 30 degreees for 16h.
4. Dilute it 50 folds with the same media and incubate until OD550 is about 0.15.
5. Ditribute 3ml of the culture to falcon tubes and add certain amount of IPTG.
6. Incubate the cultures at 30 degrees and measure OD550 of them every 30 min.

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