Template:Kyoto/Notebook/Sep 9

From 2013.igem.org

Contents

Sep 9

PCR

Nakagawa

KaiA(plasmid)10x bufferdNTPsMgSO4Primer(661-RBS-KaiA-fwd)Primer(kaiA-662-rev-ver2)Kod-plus ver2MilliQtotal
1.02.52.51.50.750.750.515.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C58.5°C68°C--
2min10s30s30s30cycles
KaiB(plasmid)10x bufferdNTPsMgSO4Primer(661-RBS-KaiA-fwd-ver2)Primer(kaiB-663-rev-ver2)Kod-plus ver2MilliQtotal
1.02.52.51.50.750.750.515.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C58.5°C68°C--
2min10s30s30s30cycles
P-KaiBC(PCR products)10x bufferdNTPsMgSO4Primer(prefix-P-kaiBC-fwd-ver2)Primer(P-KaiBC-suffix-rev-verw)Kod-plus ver2MilliQtotal
0.72.52.51.50.750.750.515.825
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C58.5°C68°C--
2min10s30s30s30cycles
DT(plasmid)10x bufferdNTPsMgSO4Primer(664-DT-fwd-ver2)Primer(DT-suffix-660-rev-ver2)Kod-plus ver2MilliQtotal
1.02.52.51.50.750.750.515.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C58.5°C68°C--
2min10s30s30s30cycles

Gel Extraction

Yoshida

LaneDNAEnzyme
1100bp ladder--
2KaiA--
3
4KaiB--
5
6P-KaiBC--
7
8DT--
9
10NC--
11100bp ladder--

Igku 992.jpg
Igku 993.jpg

Nameconcentration[µg/mL]260/280260/230
KaiC28.51.2611.92
Plac53.91.821.61
KaiB59.61.612.20
KaiB76.51.861.44
DT98.71.471.43

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
9/8 pSB1C383.21.450.76
9/8 RBS-lysis2-DT72.81.670.97
9/8 spinach-DT152.61.881.97
Nuclease-Free water-0.4-14.300.25

Restriction Enzyme Digestion

No name

9/9 pSB1C3XbaIPstIBufferBSAMilliQtotal
2 cuts24µL1µL1µL3µL3µL22µL30µL*
NC1.2µL0µL0µL1µL1µL6.8 µL10µL
9/9 pSB1C3XbaIPstIBufferBSAMilliQtotal
2 cuts27.4µL1µL1µL3µL3µL22µL30µL*
NC1.3µL0µL0µL1µL1µL6.7µL10µL
9/9 spinach-DTXbaIPstIBufferBSAMilliQtotal
2 cuts10.8µL1µL1µL3µL3µL22µL30µL*
NC0.5µL0µL0µL1µL1µL7.5µL10µL
  • "*"=be evaporated

Transformation

No name

NameSampleCompetent CellsTotalPlate
Pcon+RBS-luxI-DT2µL20µL22µLAmp
Pcon-RBS-luxR-DT+Plux-GFP2µL20µL22µL
Ptet-PT181 antisense+spinach-DT-Pbad/araC+RBS-lux-DT2µL20µL22µL

Ligation

No Name

stateVectorInserterLigation High ver.2
experiment9/8 F1-attenuator(XbaI&PstI)5.3µg9/8 pSB1C3(XbaI&PstI)8.9µg3.5µg
experiment9/8 F3m2-attenuator(XbaI&PstI)4.5µg9/8 pSB1C3(XbaI&PstI)8.9µg3.5µg
experiment9/8 F1-antisense(XbaI&PstI)1.7µg9/8 pSB1C3(XbaI&PstI)8.9µg3.5µg
experiment9/8 F6-antisense(XbaI&PstI)2.9µg9/8 pSB1C3(XbaI&PstI)8.9µg3.5µg
experiment9/8 aptamer12-P(EcoRI&SpeI)3.1µg9/8 pSB1C3(EcoRI&SpeI)8.8µg3.5µg
experiment9/8 aptamer12-1M(EcoRI&SpeI)3.1µg9/8 pSB1C(EcoRI&SpeI)38.8µg3.5µg

M9 medium

Shimizu & Hirano

EDTA consenlationNa2HPO4KH2PO4NaClNH4ClFe(Ⅲ)-EDTAdistilled watertotal
1mMol300mg150mg25mg50mg21mgup to 10mL10mL
10mMol300mg150mg25mg50mg210.5mgup to 10mL10mL
50mMol300mg150mg25mg50mg1052.7mgup to 10mL10mL

Colony PCR

No name

Samplebase pair
9/8 Plac-spinach-DT-(1)659
9/8 Plac-spinach-DT-(2)659
9/8 Pcon-pt181attenuator-DT-(1)584
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s40s30cycles

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder--
2Spinach-DTXbaI+PstI
3
5RBS-lacZα-DTXbaI+PstI
6

Igku 995.jpg

LaneDNAEnzyme
1100bp ladder--
3pSB1C3XbaI+PstI
4

Igku 996.jpg

Ligation

No name

stateVectorInserterLigation High ver.2
experimentpSB1C3(XbaI+PstI)xx µL9/8 F1-attenuator(XbaI+PstI)xx µLxx µL
experimentpSB1C3(XbaI+PstI)xx µL9/8 F3m2-attenuator(XbaI+PstI)xx µLxx µL

PCR

Tatsui and Yoshida

KaiC10x buffer KOD plus ver22M dNTPMgSO4Primer Mix(F&R)Kod-plus ver2MilliQtotal
0.52.52.51.51.50.516.025.0
1.02.52.51.51.50.515.525.0
2.02.52.51.51.50.514.525.0
4.02.52.51.51.50.512.525.0
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s2min30cycles
Plac(100pg)10x buffer KOD plus ver22M dNTPMgSO4Primer Mix(F&R)Kod-plus ver2MilliQtotal
0.32.52.51.51.50.516.225.0
0.62.52.51.51.50.515.925.0
1.22.52.51.51.50.515.325.0
2.42.52.51.51.50.514.125.0
0.02.52.51.51.50.516.525.0
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s2min30cycles

Liquid Culture

No name

Samplemedium
pSB2C3(master)-2Plusgrow medium(+CP)

Restriction Enzyme Digestion

No name

8/17 lysis1-2XbaIPstIBSABufferMilliQtotal
2 cuts8.1µL1µL1µL3µL3µL13.9µL30µL
NC0.4µL0µL0µL1µL1µL7.6µL10µL
8/17 lysis3-1XbaIPstIBSABufferMilliQtotal
2 cuts6.1µL1µL1µL3µL3µL15.9µL30µL
NC0.3µL0µL0µL1µL1µL7.7µL10µL
9/15 PlacXbaIPstIBSABufferMilliQtotal
1 cuts12.1µL0µL1µL3µL3µL10.9µL30µL
NC0.3µL0µL0µL1µL1µL7.7µL10µL

PCR

Yoshida

KaiA(KaiABC) 100pg/µL10x buffer KOD plus ver2dNTPsMgSO4Primer Mix(Fwd&Rev)KOD plus ver2MilliQtotal
2.02.52.51.51.50.514.525
4.02.52.51.51.50.512.525
8.02.52.51.51.50.58.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C60°C68°C--
2min10s30s1min30cycles
PKaiBC 400pg/µL10x buffer KOD plus ver2dNTPsMgSO4Primer Mix(Fwd&Rev)KOD plus ver2MilliQtotal
0.52.52.51.51.50.516.025
1.02.52.51.51.50.515.525
2.02.52.51.51.50.514.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C60°C68°C--
2m10s30s1m30cycles

Liquid Culture

No name

Samplemedium
9/2 Spinach-DTPlusgrow medium(+CP)
8/29 Plux-RBS-GFP-DTPlusgrow medium
9/6 master plate Plusgrow medium(+Amp)
3 Pcon-PT181 attenuatorPlusgrow medium

Gel Extraction

No name

LaneDNAEnzyme
11kbp ladder--
3lysis1XbaI+PstI
4
5
6

Igku 9910.jpg
Igku 9911.jpg

LaneDNAEnzyme
11kbp ladder--
3lysis3XbaI+PstI
4
5
6

Igku 9912.jpg
Igku 9913.jpg

Nameconcentration[µg/mL]260/280260/230
（´ω｀）
（ΦωΦ^）

Electrophoresis

Yoshida

LaneSampleEnzyme1Enzyme2
11kb ladder----
2Plac 100pg----
3Plac 200pg----
4Plac 400pg----
5Plac 800pg----
6NC----
71kbp ladder----

Igku 9914.jpg

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