Team:Tokyo Metropolitan/Project/Fiber/Protocol

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E.coli Fiber Project Protocol

Contents 1 Grow up a culture of A.xylinum in media 2 Grow up a culture of E.coli 3 Direct PCR 4 Electrophoresis 5 DNA purification from agarose gel with QIAGEN 6 Digestion 7 Ligation 8 Transformation 9 Miniprep (extraction of plasmid kit)

Grow up a culture of A.xylinum in media

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 　 Prepare media as outlined (add the materials as above) 　 Autoclave to sterilize media(121°C　20minute). 　 Streak/inoculate A.xylinum onto plates or in media. 　 Incubate cells at 28°C for 2-3 days. 　 If using a freeze dried source of A.xylinum, growth may take up to 4 days. Note 　 The growth of A.xylinum does not give a cloudy appearance in the media, the media will remain transparent to slightly translucent in appearance. 　 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. 　 A.xylinum will grow well at room temperature in aerobic conditions.

Equipment 　

• autoclave 　
• incubator 　
• scale 　
• bunsen burner 　
• flask(1l or500ml) 　
• plate 　
• spreader 　
• pipet 　
• pipet 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.

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

Direct PCR

Material a colony 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 Add and mix above materials to 50μl in each PCR tubes on the ice Pick up E.coli K12 cells from its colony and add into the PCR tubes Setting tips and elongation in the thermal cycler initialization :95°C 3min denaturation:96°C 1min annealing :55°C 5min elongation:72°C 1min (30cycles from 96°C 1min to 72°C 1min) reaction stop :10°C

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 Measure out agarose into a beaker with the 300ml of TAE buffer.Microwave until the agarose is fully melted(5minutes×3). 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. 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. 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. 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. set agarose gel and add TAE buffer in gel box. mix DNA and Loading buffer and then put in well them(marker sets another well). load DNA at 100V for two third of entire(about 15minutes). image the consequence of electrophoreses.

DNA purification from agarose gel with QIAGEN

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 cut gel of electrophoreses add pieces of gel to tubes take 300µl QG buffer into tubes and dissolve at 50°C add a solution of QG buffer and gel to tubes for column centrifuge 15000rpm/1min throw “flow-thru” away and take 700µl PE buffer centrifuge 15000rpm/1min throw flow-thru away centrifuge 15000rpm/1min change tube for column add 50µl of EB buffer (aim to center of tube) centrifuge 15000rpm/1min

Digestion

Material DNA for digestion 50µl 10×M buffer 5µl XbaI 1µl SpeI 1µl 　Equipment incubater PCR tube pipet freezer freezer box 　Procedure add above materials to a tube Incubate 37°C for 2~16hours

Ligation

Material 2×ligation Mix(Nippon gene) plasmid DNA insert DNA 　Equipment incubater PCR tube pipette freezer freezer box 　Procedure Incubate at room temperature for 5minutes

Transformation

Material E.coli Competent cell (JM109) ligation production(refer to Ligation) Equipment autoclave incubator heater bunsen burner flask(500ml) plate tube pipette pipette tip ice Procedure add 50µl of E.coli Competent cells to tubes which was used ligation incubate the cells on ice for 30minutes heat shock the cells by heater at42°C for 45sec incubate the cells on ice for 2 min streak the cells on LB medium plate added chloramphenicol incubate cells at 37°C during for 14hours

Miniprep (extraction of plasmid kit)

Material E.coli (plasmid) SolutionⅠ SolutionⅡ SolutionⅢ matrix TE buffer Equipment centrifuge microwave vortex mixer tube filter Procedure pick E.coli cells up from a colony and add to 2ml tube centrifuge 15000rpm/30sec and throw supernatant fluid away add 120µl of Solution I and vortex add 250µl of Solution II and shake with hand add 250µl of Solution III centrifuge 15000rpm/5min set spin filter in 2ml tube add supernatant to spin filter, then add 200µl of matrix and suspend with pipet centrifuge 15000rpm/30sec and throw supernatant fluid away add wash buffer 500µl and then centrifuge 15000rpm/30sec and throw supernatant fluid away (twice) centrifuge 15000rpm/2min and throw supernatant fluid away set spin filter in 1.5ml tube add TE buffer to 15ml falcon tube and heat 15min with microwave add 100µl of TE buffer centrifuge 15000rpm/1min