• 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, 15000 rpm for 1 min
3. Discarded the supernatant and added remaining solution.
4. Centrifuged at 4C, 15000 rpm for 1 min
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, 13000 rpm for 10 min
10. Transfered the supernatant to filtration column
11. Centrifuged at 4C, 13000 rpm for 1 min
12. Discarded the flow-through
13. added 500 uL of Buffer PB to filtration column
14. Centrifuged at 4C, 13000 rpm for 1 min
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, 13000 rpm for 1 min
19. Stored at -20C

Colony PCR of AraC+RBS+pSB1A3

• Selected 16 colonies at random, and PCRed them.
• PCR mix used is shown in the table bellow

Result of colony PCR

• electrophoresed the samples

1 lane -> Marker λ/HindIII EcoRI 5 uL

2~8 lane -> colony No.1~7

9 lane -> Marker λ/HindIII EcoRI 5 uL

10~16 lane -> colony No.8~14

• Colony No.1 will have the plasmid,so we put the sample into 2 mL LB added 2 uL Ampicillin(100 ug/uL).Incubated it in a shaker at 37C, 180 rpm.