Team:HokkaidoU Japan/Notebook/September23


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(Colony PCR of AraC+RBS+pSB1A3)
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*Amplifiable BAC plasmid ([ BBa_J61031]) purification
*Amplifiable BAC plasmid ([ BBa_J61031]) purification

Revision as of 08:31, 27 October 2010

  • 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

Colony PCR of AraC+RBS+pSB1A3

  1. Selected 16 colonies at random
  2. Suspended in 10 uL H2O in 0.5 mL tubes
  3. PCR mix used is shown in the table bellow
Reagent Amount
Taq Master Mix 90 uL
Ex-F 1.8 uL
Ps-R 1.8 uL
Total 93.6 uL