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Team:HokkaidoU Japan/Notebook/September23
From 2010.igem.org
(Difference between revisions)
| Line 11: | Line 11: | ||
#Transfered 1.3 mL of BAC plasmid solution incubated overnight into 1.5 mL tube | #Transfered 1.3 mL of BAC plasmid solution incubated overnight into 1.5 mL tube | ||
| - | #Centrifuged at 4C, | + | #Centrifuged at 4C, 15000 rpm for 1 min |
#Discarded the supernatant and added remaining solution. | #Discarded the supernatant and added remaining solution. | ||
| - | #Centrifuged at 4C, | + | #Centrifuged at 4C, 15000 rpm for 1 min |
#Discarded the supernatant | #Discarded the supernatant | ||
#Suspended on 250 uL of Buffer P1 | #Suspended on 250 uL of Buffer P1 | ||
#Added 250 ul Buffer P2 inverted few times to mix, solution turned green | #Added 250 ul Buffer P2 inverted few times to mix, solution turned green | ||
#Added 350 ul Buffer N3 mixed by inversion, precipitation apeared | #Added 350 ul Buffer N3 mixed by inversion, precipitation apeared | ||
| - | #Centrifuged at 4C, | + | #Centrifuged at 4C, 13000 rpm for 10 min |
#Transfered the supernatant to filtration column | #Transfered the supernatant to filtration column | ||
| - | #Centrifuged at 4C, | + | #Centrifuged at 4C, 13000 rpm for 1 min |
#Discarded the flow-through | #Discarded the flow-through | ||
#added 500 uL of Buffer PB to filtration column | #added 500 uL of Buffer PB to filtration column | ||
| - | #Centrifuged at 4C, | + | #Centrifuged at 4C, 13000 rpm for 1 min |
#Discarded the flow-through centrifuged for 1min to remove remaining buffer | #Discarded the flow-through centrifuged for 1min to remove remaining buffer | ||
#Transfered filtration column to a new 1.5 ml tube | #Transfered filtration column to a new 1.5 ml tube | ||
#Resuspended on 50 ul of TE and incubated at RT for 1min | #Resuspended on 50 ul of TE and incubated at RT for 1min | ||
| - | #Centrifuged at 4C, | + | #Centrifuged at 4C, 13000 rpm for 1 min |
| - | #Stored at - | + | #Stored at -20C |
== Colony PCR of AraC+RBS+pSB1A3 == | == Colony PCR of AraC+RBS+pSB1A3 == | ||
#Selected 16 colonies at random | #Selected 16 colonies at random | ||
| - | #Suspended in 10 uL | + | #Suspended in 10 uL DW in 0.5 mL tubes |
#PCR mix used is shown in the table bellow | #PCR mix used is shown in the table bellow | ||
| Line 41: | Line 41: | ||
|- | |- | ||
|Taq Master Mix | |Taq Master Mix | ||
| - | | | + | |80 uL |
|- | |- | ||
|Ex-F | |Ex-F | ||
| - | |1. | + | |1.6 uL |
|- | |- | ||
|Ps-R | |Ps-R | ||
| - | |1. | + | |1.6 uL |
|- | |- | ||
|style="border-top:1px solid #996;"|'''Total''' | |style="border-top:1px solid #996;"|'''Total''' | ||
|style="border-top:1px solid #996;"|'''93.6 uL''' | |style="border-top:1px solid #996;"|'''93.6 uL''' | ||
|} | |} | ||
Revision as of 08:34, 27 October 2010
since 13 Jul 2010
since 1 Aug 2010
- 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
- Transfered 1.3 mL of BAC plasmid solution incubated overnight into 1.5 mL tube
- Centrifuged at 4C, 15000 rpm for 1 min
- Discarded the supernatant and added remaining solution.
- Centrifuged at 4C, 15000 rpm for 1 min
- Discarded the supernatant
- Suspended on 250 uL of Buffer P1
- Added 250 ul Buffer P2 inverted few times to mix, solution turned green
- Added 350 ul Buffer N3 mixed by inversion, precipitation apeared
- Centrifuged at 4C, 13000 rpm for 10 min
- Transfered the supernatant to filtration column
- Centrifuged at 4C, 13000 rpm for 1 min
- Discarded the flow-through
- added 500 uL of Buffer PB to filtration column
- Centrifuged at 4C, 13000 rpm for 1 min
- Discarded the flow-through centrifuged for 1min to remove remaining buffer
- Transfered filtration column to a new 1.5 ml tube
- Resuspended on 50 ul of TE and incubated at RT for 1min
- Centrifuged at 4C, 13000 rpm for 1 min
- Stored at -20C
Colony PCR of AraC+RBS+pSB1A3
- Selected 16 colonies at random
- Suspended in 10 uL DW in 0.5 mL tubes
- PCR mix used is shown in the table bellow
| Reagent | Amount |
|---|---|
| Taq Master Mix | 80 uL |
| Ex-F | 1.6 uL |
| Ps-R | 1.6 uL |
| Total | 93.6 uL |
