Team:HokkaidoU Japan/Notebook/September23

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  • Amplifiable BAC plasmid (BBa_J61031) purification
  • Colony PCR of AraC+RBS+pSB1A3
  • Electroporetion of BAC plasmid into DH5α MG1655

Bac Vecter Purification

Used Qiagen miniprep kit, qiaprep for miniprep

  1. Transfered 1.3 mL of BAC plasmid solution incubated overnight into 1.5 mL tube
  2. Centrifuged at 4C, 15000rpm for 1min
  3. Discarded the supernatant and added remaining solution.
  4. Centrifuged at 4C, 15000rpm for 1min
  5. Discarded the supernatant
  6. Suspended on 250 uL of Buffer P1
  7. Added 250 ul Buffer P2 inverted few times to mix, solution turned green
  8. Added 350 ul Buffer N3 mixed by inversion, precipitation apeared
  9. Centrifuged at 4C, 13000rpm for 10min
  10. Transfered the supernatant to filtration column
  11. Centrifuged at 4C, 13000rpm for 1min
  12. Discarded the flow-through
  13. added 500 uL of Buffer PB to filtration column
  14. Centrifuged at 4C, 13000rpm for 1min
  15. Discarded the flow-through centrifuged for 1min to remove remaining buffer
  16. Transfered filtration column to a new 1.5 ml tube
  17. Resuspended on 50 ul of TE and incubated at RT for 1min
  18. Centrifuged at 4C, 13000rpm for 1min
  19. Stored at -20


AraC+RBS+pSB1A3のコロニーPCR

  1. コロニーを無作為に16個選び、番号を振った。
  2. 0.5 mLチューブを16本用意し、それぞれにDWを10 uLずつ分注した。
  3. チューブに番号を振り、対応するコロニーを懸濁した。
  4. コロニーPCR溶液を以下の組成に従って調整した。
Reagent Amount
Taq Master Mix 90 uL
Ex-F 1.8 uL
Ps-R 1.8 uL
Total 93.6 uL