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E.coli Fiber Project Protocol

Contents 1 　Grow up a culture of A.xylinus in media 1.1 　Grow up a culture of A.xylinus in media recommend by Open Wet Ware 1.2 　Grow up a culture of A.xylinus in media recommend by JCM 2 　Grow up a culture of E.coli 3 　Direct PCR 4 　Electrophoreses PCR productions 5 　DNA purification from agarose gel with QIAGEN

　Grow up a culture of A.xylinus in media

　Grow up a culture of A.xylinus in media recommend by Open Wet Ware

Material 　 A.xylinus JCM strain 7664 　 500 ml of liquid Acetobacter media(Open Wet Ware recommended) 　　 Glucose - 1.0 g 　　 Peptone - 2.5 g 　　 Yeast extract - 2.5 g 　　 Na2HPO4 - 1.35 g 　　 Citric acid - 0.75 g 　　 Distilled water - 500 ml 　(If you are making plates, use the same protocol but add 7.5 g of agar.) Equipment 　 autoclave 　 incubator 　 scale 　 bunsen burner 　 flask(1l or500ml) 　 plate 　 spreader 　 pipet 　 pipet tip Procedure 　 Prepare media as outlined (add the materials as above) 　 Autoclave to sterilize media(121°C　20minute). 　 Streak/inoculate A.xylinus onto plates or in media. 　 Incubate cells at 26°C for 2-3 days. 　 If using a freeze dried source of A.xylinus, growth may take up to 4 days. Note 　 The growth of A.xylinus does not give a cloudy appearance in the media, the media will remain transparent to slightly translucent in appearance. 　 The growth of A.xylinus 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.xylinus will grow well at room temperature in aerobic conditions.

　Grow up a culture of A.xylinus in media recommend by JCM

Material 　 A.xylinus JCM strain 7664 　 500 ml of liquid Acetobacter media(Open Wet Ware recommended) 　　 Glucose - 100g 　　 Yeast extract - 10g 　　 CaCO3 30g 　　 Distilled water - 1000 ml 　　 　(If you are making plates, use the same protocol but add 7.5 g of agar.) Equipment 　 autoclave 　 incubator 　 scale 　 bunsen burner 　 flask(1l or500ml) 　 plate 　 spreader 　 pipet 　 pipet tip Procedure 　 Prepare media as outlined (add the materials as above) 　 Autoclave to sterilize media(121°C　20minute). 　 Streak/inoculate A.xylinus onto plates or in media. 　 Incubate cells at 26°C for 2-3 days. 　 If using a freeze dried source of A.xylinus, growth may take up to 4 days. Note 　 The growth of A.xylinus does not give a cloudy appearance in the media, the media will remain transparent to slightly translucent in appearance. 　 The growth of A.xylinus 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.xylinus will grow well at room temperature in aerobic conditions.

　Grow up a culture of E.coli

Material E.coli K12strain 1l of solid LB media Distilled water 1l LB agar (from Becton, Dickinson and Company) 35g 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 E.coli K12 colony 10µM/l forward Primer 5µl 10µM/l reverse Primer 5µl 10×EX taq buffer 10µl 2.5mM each dNTP mixture 10µl EX taq polymerase 1µl milli-Q 71µl Equipment thermal cycler vortex mixer PCR-tubes pipet pipet 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

　Electrophoreses PCR productions

Material Equipment Procedure

　DNA purification from agarose gel with QIAGEN

Material 　　QIAGEN(gel extraction kit) 　　-QG buffer 300µl 　　-PE buffer700µl 　　-EB buffer 50µl 　　-tube for column 　Equipment 　　centrifuge 　　heating plate 　　pipet 　　pipet 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