Team:Tokyo Metropolitan/Project/Fiber/Protocol

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E.coli Fiber Project Protocol

Contents

 Grow up a culture of A.xylinus in media

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

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

 Grow up a culture of E.coli

 Direct PCR

 Electrophoreses PCR productions

 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