Team:HokkaidoU Japan/Notebook/September23

• Amplifiable BAC plasmid ([http://partsregistry.org/Part:BBa_J61031 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