Team:HokkaidoU Japan/Notebook/September30
From 2010.igem.org
- Digestion and Ligation of Arabinose Promoter,T3SSsignal and pSB1T3
- Transformation
parts information
Arabinose Promoter 1210 bp(+primer:1259 bp)
3-20B pSB2K3
PCRed in 21 September
RBS+T3SSsignal 583 bp(+primer:636 bp)
PCRed in 29 September
pSB1T3 2463 bp(+primer:2504 bp)
PCRed in 26 August
Digestion and Ligation of Arabinose Promoter,T3SSsignal and pSB1T3
digestion mix
Reagent | Amount |
---|---|
10x H buffer | 2 uL |
DW | 1.6 |
pSB1T3 | 1 |
BSA | 2 |
EcoR I | 0.2 |
Pst I | 0.2 |
Total | 20 uL |
Reagent | Amount |
---|---|
10x H buffer | 2 uL |
DW | 11.7 |
Promoter | 3.5 |
BSA | 2 |
EcoR I | 0.5 |
Spe I | 0.3 |
Total | 20 uL |
Reagent | Amount |
---|---|
10x M buffer | 5 uL |
DW | 32.8 |
T3SSsignal | 2.5 |
BSA | 5 |
Xba I | 4.5 |
Pst I | 0.2 |
Total | 50 uL |
- incubated at 37C for 1h
- electrophoresed 3 samples with 6 uL λ/HindIII EcoR I
- extracted 3 samples from a gel
- added 4.5 uL of 3 M CH3COONa
- added 125 uL 100% EtOH
- centrifuged at 4C,15000 rpm for 5 min
- discarded the supertenant
- added 100 uL 70% EtOH
- centrifuged at 4C,15000 rpm for 5 min
- dried up the samples
- dissolved the samples with 2 uL of TE
- mixed the 3 samples
- added 6 uL Mighty Mix (Ligation Kit)
- incubated at 16C for 30 min
- put the sample into 100 uL competent cell
- incubated at 0C for 30 min
- heatshocked at 42C for 60 sec
- incubated at 0C for 5 min
- added 400 uL of SOB
- incubated at 30C for 1.5 hrs
- incubated at 37C for 45 min
- plated the sample on LBT medium