Team:HokkaidoU Japan/Notebook/September30
From 2010.igem.org
(Difference between revisions)
Line 8: | Line 8: | ||
<u>parts information</u> | <u>parts information</u> | ||
- | Arabinose Promoter | + | Arabinose Promoter 1210 bp(+primer:1259 bp) |
3-20B pSB2K3 | 3-20B pSB2K3 | ||
Line 15: | Line 15: | ||
- | RBS+T3SSsignal | + | RBS+T3SSsignal 583 bp(+primer:636 bp) |
PCRed in 29 September | PCRed in 29 September | ||
- | pSB1T3 | + | pSB1T3 2463 bp(+primer:2504 bp) |
PCRed in 26 August | PCRed in 26 August | ||
- | + | = Digestion and Ligation of Arabinose Promoter,T3SSsignal and pSB1T3 = | |
- | + | ==digestion mix== | |
{|style="text-align:center; float:left;" class="protocol" | {|style="text-align:center; float:left;" class="protocol" | ||
!Reagent | !Reagent | ||
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<div style="clear:both"></div> | <div style="clear:both"></div> | ||
- | # incubated at 37C for | + | # incubated at 37C for 1h |
- | # electrophoresed 3 samples with 6 uL λ/''Hin'' | + | # electrophoresed 3 samples with 6 uL λ/''Hin''dIII EcoR I |
# extracted 3 samples from a gel | # extracted 3 samples from a gel | ||
- | # added 4.5 uL of | + | # added 4.5 uL of 3 M CH<sub>3</sub>COONa |
# added 125 uL 100% EtOH | # added 125 uL 100% EtOH | ||
# centrifuged at 4C,15000 rpm for 5 min | # centrifuged at 4C,15000 rpm for 5 min |
Revision as of 08:51, 27 October 2010
- 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