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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 　1l of liquid Acetobacter media(JCM recommended) 　　-Glucose - 100g 　　-Yeast extract - 10g 　　-CaCO3 30g 　　-Distilled water - 1000 ml 　(If you are making plates, use the same protocol but add 15g 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
Equipment
Procedure

　Direct PCR

Material
Equipment
Procedure

　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