Team:Tokyo Metropolitan/Project/Fiber/Protocol

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E.coli Fiber Project Protocol

Contents 1 Protocol1:Grow up a culture of A.xylinum 2 Protocol2:Grow up a culture of E.coli 3 Protocol3:PCR 4 Protocol4:Agarose gel electrophoresis 5 Protocol5:DNA extraction from agarose gel 6 Protocol6:DNA Purification with silica gel 7 Protocol7:Restriction enzyme digestion 8 Protocol8:Ligation 9 Protocol9:Transformation 10 Protocol10:Extraction of plasmid 11 Protocol11:Sequence

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 　 Prepare media as outlined (add the materials as above) 　 Autoclave to sterilize medium(121°C　20minute). 　 Streak/inoculate A.xylinum onto plates or in media. 　 Incubate cells at 28°C for 2-3 days. 　 Note 　 The growth of A.xylinum does not give a cloudy appearance in the medium, the medium 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.

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 Prepare medium as outlined (add the materials as above). Autoclave to sterilize medium(121°C　20minute). Streak/inoculate E.coli onto plates or in medium. 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 Add and mix above materials to each PCR tubes on the ice Add templete DNA (or cells) into the PCR tubes Setting PCR tubes in the thermal cycler 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 Measure out agarose into a beaker with TAE buffer and 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.

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 Cut a band of DNA in agarose gel Add pieces of gel to tubes Take 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-through away and take PE buffer Centrifuge 15000rpm/1min Throw flow-through away Centrifuge 15000rpm/1min Change tube for column Add EB buffer (aim to center of tube) 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 Add above materials to 50μl 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 Add materials to 20μl 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 Mix 50µl of E.coli Competent cells and DNA incubate the cells on ice for 30minutes heat shock the cells at 42°C for 45sec incubate the cells on ice for 2 min Add 4 times volume of SOC broth streak the cells on LB medium plate added anti-biotic 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 Add 2ml of preculture of E.coli into a tube centrifuge 15000rpm/30sec and throw supernatant fluid away add 120µl of resuspention solution and vortex add 250µl of lysis Solution and shake with hand add 250µl of neutralization　Solution and shake with hand centrifuge 15000rpm/5min set spin filter in 2ml tube add supernatant to spin filter, then add 200µl of quantum prep mix and suspend with pipette 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 15sec with microwave add 100µl of TE buffer 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 mix DNA(<50ng),Big Dye,primer,ethanol and Distilled water PCR Add EDTA and Ethanol and put at room temperature　(15min) Centrifuge (15000rpm,30min) and throw away supernatant Add ethanol again Centrifuge (15000rpm,15min) and throw away supernatant Add Hi-Di solution Heat at 95℃ Transfer these sample to plate for sequence Read sequence