Team:Kyoto/MethodAndMaterial
From 2012.igem.org
Contents |
Protocol
Western blotting
Gel solution
Running gel | Stacking gel | |
---|---|---|
1.5M Tris-HCl(pH8.8) | 2.5mL | - |
0.25M Tris-HCl(pH6.8) | - | 3.0mL |
30% Acrylamide | 5mL | 0.6mL |
10% SDS | 0.2mL | 0.12mL |
DW | 2.3mL | 2.3mL |
TEMED | 15µL | 7µL |
10% APS | 100µL | 60µL |
Total | 10mL | 6mL |
4x Sample buffer
1M Tris-HCl | 2mL |
SDS | 0.8g |
100% glycerol | 4mL |
14.7M mercaptoethanol | 0.4mL |
0.5M EDTA | 1mL |
Bromophenol blue | 8.0mg |
DW | 2.6mL |
10x electrode buffer
Tris | 15.15g |
Glycin | 71.55g |
10%SDS | 50mL |
DW | 450mL |
Total | 500mL |
blotting buffer
Tris | 12g |
Glycin | 14.4g |
DW | 800mL |
Methanol | 200mL |
TBST
50mM | Tris |
150mM | NaCl |
0.1% | Tween-20 |
blocking buffer
- TBST
- 5% skim milk
AP color development buffer
- 100mM Tris (pH8.5)
- 100mM NaCl
- 5mM MgCl2
- Spin down 100µL culture and suspend into sample buffer.
- Boil at 95°C for 10 min.
- Apply 10µL to polyacrylamide gel.
- Electrophorese at 500V, 30mA for 50 min.
- Transfer at 50V, 100mA for 30 min.
- Add blocking buffer to the membrane and incubate with shaking for 30 min. at room temperature.
- Add appropriate primary antibody diluted in TBST and incubate with shaking for 30 min. at RT.
- Wash with TBST and incubate with shaking for 10 min, two times.
- Add secondary antibody conjugated with AP and incubate with shaking for 30 min. at RT.
- Wash with TBST and incubate with shaking for 10 min, three times.
- Add 66µL NBT and 33µLof BCIP into color development buffer., and add the mixture to the membrane.
- After the coloring, wash with DW.
Verification of R9 function by GFP
Verification of R9 function by TAKEUCHI
R9(20µg/µL) | 0.9µL |
GFP(1.2mg/mL) | 2.23µL |
RBS | 16.85µL |
total | 20µL |
X5
Method:
1. Peel cuticles on parafilm by using the head of pencil.(Menasha,wI,54952)
2. Put plant cells into GFP&R9 or GFP for 5~30min.
3. Put plant cells into PBS.
4. Hoechst dyeing.
1 | 2 | 3 | 4 | 5 | 6 | |
R9 | o | o | o | x | o | o |
cuticle | o | o | o | o | x | x |
soak in GFP | 5min | 15min | 30min | 5min | 5min | 30min |
<groupparts>iGEM012 Kyoto</groupparts>
Strains
E.coli
JM109 endA, recA1, gyrA96,thi,hsdR17,(rκ-,mκ+),relA1, supE44,λ-,Δ(lac-proAB),[F',traD36,proAB,laclqZΔM15]
BL21(DE3) F-, ompT, hsdSB(rB -mB - ), gal, dcm, (DE3)
Plant
Arabidopsis thaliana ecotype Columbia
Primers
Florigen
Name | sequence |
---|---|
FT(RBS+)forward | gctctagagaaagaggagaaaGTCGACATGTCTATAAATATAAGAGACCCTCT |
FT(RBS+)reverse | aaactgcagcggccgctactagtaCCTTGAGCTCCTAAAGTCTTCTTCCTC |
FT(RBS-)forward | ggaattcgcggccgcttctagagATGTCTATAAATATAAGAGACCCTCTT |
FT for sequence forward | TGACCATGATTACGCCAAGC |
FT for sequence reverse | ACGGCCAGTGAATTGTAATACG |
FT(+His)forward | TCTATAAATATAAGAGACCCTCTTATAGTAAGCAGAGTTG |
FT(+His)reverse | ATGGTGATGGTGATGGTGCATGTCGACtttctcctctttctctagtatctcc |
FT mutation forward | CATTCATCGTGTCGTGTTTATATTGTTTCG |
FT mutation reverse | CCAGCAGTGGGACTTGGATTTTCGTAACAC |
FT mutation forward2 | CATTCATCGTGTCGTGTTTATATTGTTTCGACAGC |
TUBULIN forward | AAACTCACTACCCCCAGCTTTG |
TUBULIN reverse | CACCAGACATAGTAGCAGAAATCAAGT |
SEP3 forward | CTAAGACTAAGGTTAGCTGATGGGTA |
SEP3 reverse | ATGATGACGACCGTAGTGATCAA |
FUL forward | TGCGTAACCTCCTCCAGAGAT |
FUL reverse | GTTCTACTCGTTCGTAGTGGTAGGAC |
AP1 forward | ACATCCGCACTAGAAAAAACCAAC |
AP1 reverse | CTTGGTTCTGCTGATCCCACA
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