Team:Tokyo Metropolitan/Project/Fiber/Protocol

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E.coli Fiber Project Protocol

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Contents

Protocol1:Grow up a culture of A.xylinum

Material 　
  • A.xylinum JCM strain 7664 　
  • liquid Acetobacter media(Open Wet Ware recommended) 　　
    • Glucose 　　
    • Peptone 　　
    • Yeast extract 　　
    • Na2HPO4 　　
    • Citric acid 　　
    • Distilled water
　(If you are making plates, use the same protocol but add agar.)

Equipment 　
  • autoclave 　
  • incubator 　
  • scale 　
  • bunsen burner 　
  • flask(1l or500ml) 　
  • plate 　
  • spreader 　
  • pipette 　
  • pipette tip

Procedure 　
  1. Prepare media as outlined (add the materials as above) 　
  2. Autoclave to sterilize media(121°C　20minute). 　
  3. Streak/inoculate A.xylinum onto plates or in media. 　
  4. Incubate cells at 28°C for 2-3 days. 　
  5. If using a freeze dried source of A.xylinum, growth may take up to 4 days.

Note 　
  1. The growth of A.xylinum does not give a cloudy appearance in the media, the media will remain transparent to slightly translucent in appearance. 　
  2. The growth of A.xylinum is accompanied by the formation of a thick cellulose matrix within the media that must be removed before cells can be pelleted for a miniprep procedure. Simply vortex briefly to break up the cellulose into chunks and remove the cellulose chunks from the media with a pipette while carefully avoiding the removal of cells. 　
  3. A.xylinum will grow well at room temperature in aerobic conditions.


Protocol2:Grow up a culture of E.coli

Material
  • E.coli K12strain
  • solid LB media
    • Distilled water
    • LB agar (Becton, Dickinson and Company)

Equipment
  • autoclave
  • incubator
  • scale
  • bunsen burner
  • flask(500ml)
  • plate
  • inoculating loop

Procedure
  1. Prepare media as outlined (add the materials as above).
  2. Autoclave to sterilize media(121°C　20minute).
  3. Streak/inoculate E.coli onto plates or in media.
  4. Incubate cells at 37°C.

Protocol3:PCR

Material
  • template DNA
  • forward Primer
  • reverse Primer
  • 10×PCR buffer
  • 2.5mM each dNTP mixture
  • DNA polymerase (Pho poly/KOD poly/Taq poly)
  • milli-Q

Equipment
  • thermal cycler
  • vortex mixer
  • PCR-tubes
  • pipette
  • pipette tip

Procedure
  1. Add and mix above materials to 50μl in each PCR tubes on the ice
  2. Pick up E.coli K12 cells from its colony and add into the PCR tubes
  3. Setting tips and elongation in the thermal cycler
    • initialization
    • denaturation
    • annealing
    • elongation

      (denaturation～elongation　30cycles)

    • reaction stop

Protocol4:Agarose gel electrophoresis

Material
  • 1% agarose gel
    • agarose S
    • TAE buffer
    • ethidium bromide
  • 1×TAE buffer
  • 10×Loading buffer
  • template DNA(PCR production)

Equipment
  • microwave
  • gel box
  • flask(500ml)

Procedure
  1. Measure out agarose into a beaker with the 300ml of TAE buffer.Microwave until the agarose is fully melted(5minutes×3).
  2. Let the agarose cool on your bench until touching the bottom of the beaker with your bare hand doesn't burn you (~5 minutes for a 50mL gel). At this point add your DNA stain, e.g., ethidium bromide. The beaker will cool unevenly (surface first), so you must be careful not to cause ripples and bubbles.
  3. While the solution is cooling, seal the open edges of your gel box with one long piece of masking tape on each side. Make sure it is sealed well or the gel will leak.
  4. Pour the agarose solution into the taped gelbox. Carefuly pop or shove to the side any bubbles, put in the comb, and let it cool for about 30 minutes, until the gel is solid.
  5. If your gel is at all purple, and you are using ethidium bromide as the DNA stain, you need to decrease your concentration by at least a factor of ten.
  6. set agarose gel and add TAE buffer in gel box.
  7. mix DNA and Loading buffer and then put in well them(marker sets another well).
  8. load DNA at 100V for two third of entire(about 15minutes).
  9. image the consequence of electrophoreses.

Protocol5-1:DNA purification from agarose gel

　Material
  • QIAGEN(gel extraction kit)
    • QG buffer 300µl
    • PE buffer 700µl
    • EB buffer 50µl
    • tube for column

　Equipment
  • centrifuge
  • heating plate
  • pipette
  • pipette tip

　Procedure
  1. cut gel of electrophoreses
  2. add pieces of gel to tubes
  3. take 300µl QG buffer into tubes and dissolve at 50°C
  4. add a solution of QG buffer and gel to tubes for column
  5. centrifuge 15000rpm/1min
  6. throw “flow-thru” away and take 700µl PE buffer
  7. centrifuge 15000rpm/1min
  8. throw flow-thru away
  9. centrifuge 15000rpm/1min
  10. change tube for column
  11. add 50µl of EB buffer (aim to center of tube)
  12. centrifuge 15000rpm/1min

Protocol5-2:DNA Purification with silica gel

Material

  • Binding buffer
  • silica gel
  • wash buffer
  • TE buffer

Equipment

  • centrifuge
  • vortex
  • aspirator
  • pipette
  • pipette tip

Procedure

  1. Add 3 times Binding buffer than digestion production
  2. Add 10µl of silica gel and mix with Vortex
  3. Centrifuge 1min
  4. Remove supernatant with aspirator
  5. Add Wash buffer and mix with vortex
  6. Centrifuge 30sec
  7. Remove supernatant with aspirator
  8. Remove ethanol by drying
  9. Add TE buffer and mix with Vortex
  10. Centrifuge 30sec
  11. DNA is solved in Supernatant

Protocol6:Restriction enzyme digestion

　Material
  • DNA for digestion 50µl
  • 10×M buffer 5µl
  • XbaI 1µl
  • SpeI 1µl

　Equipment
  • incubater
  • PCR tube
  • pipette
  • freezer
  • freezer box

　Procedure
  1. add above materials to a tube
  2. Incubate 37°C for 2~16hours

Protocol7:Ligation

　Material
  • 2×ligation Mix(Nippon gene)
  • plasmid DNA
  • insert DNA

　Equipment
  • incubater
  • PCR tube
  • pipette
  • freezer
  • freezer box

　Procedure
  1. Incubate at room temperature for 5minutes

Protocol8:Transformation

Material
  • E.coli Competent cell (JM109/NovaBlue)
  • ligation production(refer to Ligation)

Equipment
  • autoclave
  • incubator
  • heater
  • bunsen burner
  • flask(500ml)
  • plate
  • tube
  • pipette
  • pipette tip
  • ice

Procedure
  1. add 50µl of E.coli Competent cells to tubes which was used ligation
  2. incubate the cells on ice for 30minutes
  3. heat shock the cells by heater at42°C for 45sec
  4. incubate the cells on ice for 2 min
  5. streak the cells on LB medium plate added chloramphenicol
  6. incubate cells at 37°C during for 14hours

Protocol9:Miniprep (extraction of plasmid kit)

Material
  • E.coli (plasmid)
  • SolutionⅠ
  • SolutionⅡ
  • SolutionⅢ
  • matrix
  • TE buffer

Equipment
  • centrifuge
  • microwave
  • vortex mixer
  • tube
  • filter

Procedure
  1. pick E.coli cells up from a colony and add to 2ml tube
  2. centrifuge 15000rpm/30sec and throw supernatant fluid away
  3. add 120µl of Solution I and vortex
  4. add 250µl of Solution II and shake with hand
  5. add 250µl of Solution III
  6. centrifuge 15000rpm/5min
  7. set spin filter in 2ml tube
  8. add supernatant to spin filter, then add 200µl of matrix and suspend with pipet
  9. centrifuge 15000rpm/30sec and throw supernatant fluid away
  10. add wash buffer 500µl and then centrifuge 15000rpm/30sec and throw supernatant fluid away (twice)
  11. centrifuge 15000rpm/2min and throw supernatant fluid away
  12. set spin filter in 1.5ml tube
  13. add TE buffer to 15ml falcon tube and heat 15min with microwave
  14. add 100µl of TE buffer
  15. centrifuge 15000rpm/1min