Template:Kyoto/Notebook/Sep 17

From 2013.igem.org

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Latest revision as of 05:57, 26 September 2013

Contents

Sep 17

Liquid Culture

Tatsui and Hirano

Samplemedium
Spinach-DTLB medium(+CP)
Pcon-Spinach-DTLB medium(+Amp)　
Pcon-pT181 antisense-Spinach-DTLB medium(+Amp)　

PCR

Hirano

Pcon-tetR aptamer-DT(VF)BigDye5x bufferprimer(VF)MilliQtotal
1.30.51.750.50.4510.5
Pcon-tetR aptamer-DT(VR)BigDye5x bufferprimer(VR)MilliQtotal
1.30.51.750.50.4510.5
PreDenatureDenatureAnnealingExtensioncycle
96 °C96 °C50 °C60 °C--
2 min10 sec5 sec4 min30 cycles

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
11kbp ladder----
2Pcon-RBS-tetR-DTXbaIPstI
3Pcon-RBS-tetR-DT----
4pSB4K5XbaIPstI
5pSB4K5----
6pSB1C3EcoRISpeI
7pSB1C3EcoRIPstI
8pSB1C3----
9pT181 attenuatorEcoRISpeI
10pT181 attenuator----
111kbp ladder----

Igku 9171.jpg

Ligation

No Name

stateVectorInserterLigation High ver.2
experiment8/28 Plux (SpeI & PstI)5.0 µL9/13 RBS-lysis1-DT (XbaI & PstI)3.5 µL3.5 µL
experiment8/28 Plux (SpeI & PstI)5.0 µL9/13 RBS-lysis2-DT (XbaI & PstI)3.6 µL3.5 µL
experiment8/28 Plux (SpeI & PstI)11.0 µL9/13 RBS-lysis3-DT (XbaI & PstI)9.5 µL3.5 µL
experiment9/14 Plac (SpeI & PstI)2.4 µL9/16 pT181 attenuator-DT (XbaI & PstI)4.8 µL3.5 µL
experiment9/14 Plac (SpeI & PstI)2.4 µL9/3 pT181 antisense (XbaI & PstI)2.0 µL2.2 µL
experiment9/14 Plac (SpeI & PstI)2.4 µL9/10 aptamer 12_1R-DT (XbaI & PstI)1.8 µL2.1 µL
experiment9/13 pSB1C3 (XbaI & PstI)2.0 µL9/15 Pcon-PT181 antisense-Spinach-DT (XbaI & PstI)12.0 µL3.5 µL
experiment9/16 pSB1C3 (EcoRI & SpeI)1.0 µL9/15 Pcon-Spinach-DT (EcoRI & SpeI)4.1 µL2.5 µL
experiment9/16 pSB1C3 (EcoRI & SpeI)1.0 µL9/15 Pcon-RBS-tetR-DT (EcoRI & SpeI)2.0 µL1.5 µL
experiment9/16 pSB1C3 (EcoRI & SpeI)1.0 µL9/15 Pcon-PT181 attenuator-DT (EcoRI & SpeI)2.4 µL1.7 µL
experiment9/16 pSB1C3 (EcoRI & SpeI)1.0 µL9/15 Pcon-aptamer 12_1R-DT (EcoRI & SpeI)2.0 µL1.5 µL
experiment9/13 pSB1C3 (XbaI & PstI)2.0 µL9/16 Pcon-pT181-attenuator (XbaI & PstI)4.4 µL3.2 µL

Gel Extraction

No Name

LaneDNAEnzyme
11kbp ladder--
2pT181 attenuator -(2)EcoRI & SpeI
3
4

Igku 9172.jpg
Igku 9173.jpg

Nameconcentration[µg/mL]260/280260/230
pT181 attenuator (EcoI & SpeI)9.21.150.12
LaneDNAEnzyme
11kbp ladder--
2pSB1C3EcoRI & SpeI
3
4

Igku 9174.jpg
Igku 9175.jpg

Nameconcentration[µg/mL]260/280260/230
pSB1C310.01.840.40
LaneDNAEnzyme
11kbp ladder--
2pSB4K5XbaI & PstI
3
4

Igku 9176.jpg
Igku 9177.jpg

Nameconcentration[µg/mL]260/280260/230
pSB4K57.61.810.32
LaneDNAEnzyme
11kbp ladder--
2Pcon-RBS-tetR-DTXbaI & PstI
3
4

Igku 9178.jpg
Igku 9179.jpg

Nameconcentration[µg/mL]260/280260/230
Pcon-RBS-tetR-DT8.02.401.14
LaneDNAEnzyme
11kbp ladder--
2pSB1C3EcoRI & PstI
3
4

Igku 91710.jpg
Igku 91711.jpg

Nameconcentration[µg/mL]260/280260/230
pSB1C39.12.170.35

Mutation PCR

Kinose

pC KaiABC(100pg/µL)Prime STAR(2x)Primer(KaiA-BsaI MuT-Fwd)Primer(KaiA-BsaI MuT-Rev)MilliQtotal
1.025.01.01.022.050.0
1.525.01.01.021.550.0
2.025.01.01.021.050.0
PreDenatureDenatureAnnealingExtensioncycle
96 °C98 °C65 °C72 °C--
5 min10 s15 s40 s30 cycles
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