Team:Tokyo Metropolitan/Project/Fiber/Protocol

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Revision as of 07:09, 27 October 2010


E.coli Fiber Project Protocol

Spidertan.png


Contents

Protocol1:Grow up a culture of A.xylinum

Material 　
  • Acetobacter xylinum JCM 7664 strain 　
  • 1L of Acetobacter medium(From Open Wet Ware) 　　
    • Glucose:2.0g 　　
    • Peptone:5.0g 　　
    • Yeast extract:5.0g 　　
    • Na2HPO4:2.7g 　　
    • Citric acid:1.65g 　　
    • Distilled water:~1L

Equipment 　
  • autoclave 　
  • incubator 　
  • scale 　
  • bunsen burner 　
  • flask 　
  • plate(*SANPLATEC) 　
  • spreader 　
  • pipette 　
  • pipette tip

Procedure 　
  1. Prepare media as outlined (add the materials as above) 　
  2. Autoclave to sterilize medium(121°C　20minute). 　
  3. Streak/inoculate A.xylinum onto plates or in media. 　
  4. Incubate cells at 28°C for 2-3 days. 　

Note 　
  1. The growth of A.xylinum does not give a cloudy appearance in the medium, the medium 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 K12 strain
  • 1L of LB medium
    • LB agar:35g
    • Distilled water:~1L

Equipment
  • autoclave
  • incubator
  • scale
  • bunsen burner
  • flask
  • plate(*SANPLATEC)
  • inoculating loop
  • pipette
  • pipette tip

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

Protocol3:PCR

Material
  • forward Primer
  • reverse Primer
  • PCR buffer
  • dNTP mixture
  • Distilled water(milli-Q)
  • DNA polymerase
    • Pho polymerase(*Nippon gene)
    • KOD polymerase
    • Taq polymerase

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

Procedure
  1. Add and mix above materials to each PCR tubes on the ice
  2. Add templete DNA (or cells) into the PCR tubes
  3. Setting PCR tubes in the thermal cycler
  4. Cycle of PCR
    • Initialization
    • Denaturation
    • Annealing
    • Elongation

      (denaturation～elongation　30cycles)

    • Reaction stop

Protocol4:Agarose gel electrophoresis

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

Equipment
  • microwave
  • gel box
  • flask

Procedure
  1. Measure out agarose into a beaker with TAE buffer and 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:DNA extraction from agarose gel

　Material
  • QIAGEN Gel extraction kit
    • DNA in agarose gel
    • QG buffer
    • PE buffer
    • EB buffer

　Equipment
  • centrifuge
  • heating plate
  • tube with column
  • pipette
  • pipette tip

　Procedure
  1. Cut a band of DNA in agarose gel
  2. Add pieces of gel to tubes
  3. Take 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-through away and take PE buffer
  7. Centrifuge 15000rpm/1min
  8. Throw flow-through away
  9. Centrifuge 15000rpm/1min
  10. Change tube for column
  11. Add EB buffer (aim to center of tube)
  12. Centrifuge 15000rpm/1min

Protocol6: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. Supernatant contains DNA

Protocol7:Restriction enzyme digestion

　Material
  • DNA for digestion
  • 10×M buffer(*Nippon gene)
  • XbaI(*Nippon gene)
  • SpeI(*Nippon gene)
  • Distilled water

　Equipment
  • incubater
  • tube
  • pipette
  • pipette tip
  • freezer
  • freezer box

　Procedure
  1. Add above materials to 50μl
  2. Incubate 37°C for 2~16hours

Protocol8:Ligation

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

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

　Procedure
  1. Add materials to 20μl
  2. Incubate at 16℃ for 5~30minutes

Protocol9:Transformation

Material
  • E.coli Competent cell (Ecos JM109(*Nippon gene)/NovaBlue(*Merck))
  • DNA(for example ligation production)

Equipment
  • autoclave
  • incubator
  • heater
  • bunsen burner
  • plate(*SANPLATEC)
  • tube
  • pipette
  • pipette tip
  • ice

Procedure
  1. Mix 50µl of E.coli Competent cells and DNA
  2. incubate the cells on ice for 30minutes
  3. heat shock the cells at 42°C for 45sec
  4. incubate the cells on ice for 2 min
  5. Add 4 times volume of SOC broth
  6. streak the cells on LB medium plate added anti-biotic
  7. incubate cells at 37°C during for 14hours

Protocol10:Extraction of plasmid

Material
  • Preculture of E.coli
  • Bio-Rad Miniprep (extraction of plasmid kit)
    • resuspention solution
    • lysis Solution
    • neutralization　Solution
    • quantum prep mix
    • wash buffer
  • TE buffer

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

Procedure
  1. Add 2ml of preculture of E.coli into a tube
  2. centrifuge 15000rpm/30sec and throw supernatant fluid away
  3. add 120µl of resuspention solution and vortex
  4. add 250µl of lysis Solution and shake with hand
  5. add 250µl of neutralization　Solution and shake with hand
  6. centrifuge 15000rpm/5min
  7. set spin filter in 2ml tube
  8. add supernatant to spin filter, then add 200µl of quantum prep mix and suspend with pipette
  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 15sec with microwave
  14. add 100µl of TE buffer
  15. centrifuge 15000rpm/1min

Protocol11:Sequence

Material
  • DNA
  • Big Dye
  • primer
  • Distilled water
  • ethanol
  • EDTA
  • Hi-Di solution

Equipment
  • sequencer
  • centrifuge
  • vortex
  • pipette
  • pipette tip

Procedure
  1. mix DNA(<50ng),Big Dye,primer,ethanol and Distilled water
  2. PCR
  3. Add EDTA and Ethanol and put at room temperature　(15min)
  4. Centrifuge (15000rpm,30min) and throw away supernatant
  5. Add ethanol again
  6. Centrifuge (15000rpm,15min) and throw away supernatant
  7. Add Hi-Di solution
  8. Heat at 95℃
  9. Transfer these sample to plate for sequence
  10. Read sequence


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