Template:Kyoto/Notebook/Sep 13

From 2013.igem.org

(Difference between revisions)
(→Restriction Enzyme Digestion)
(→Sep 13)
Line 351: Line 351:
|experiment||9/12 Ptet-pT181 antisense (--)||1.0 µL||9/10 spinach-DT (--)||2.4 µL||3.4 µL
|experiment||9/12 Ptet-pT181 antisense (--)||1.0 µL||9/10 spinach-DT (--)||2.4 µL||3.4 µL
|}
|}
 +
 +
===PCR===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!genome DNA||KOD plus||10x buffer||dNTP||MgSO4||SasA_fwd primer||SasA_rev primer||MilliQ||total
 +
|-
 +
| ||0.5||2.5||2.5||1.5||0.75||0.75|| ||25
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|　&deg;C||　&deg;C||　&deg;C||　&deg;C||--
 +
|-
 +
|（◎Д◎）　||　||　||　||
 +
|}
 +
</div>
 +
 +
===PCR===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!genome DNA||KOD plus||10x buffer||dNTP||MgSO4||SasA_fwd primer||SasA_rev primer||MilliQ||total
 +
|-
 +
| ||0.5||2.5||2.5||1.5||0.75||0.75|| ||25
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|　&deg;C||　&deg;C||　&deg;C||　&deg;C||--
 +
|-
 +
|（◎Д◎）　||　||　||　||
 +
|}
 +
</div>
 +
 +
===Gel Extraction===
 +
<div class="experiment">
 +
<span class="author">Tatsui,Ashida</span>
 +
{| class="wikitable"
 +
!Lane||DNA||Enzyme
 +
|-
 +
|1||1kb ladder||--
 +
|-
 +
|2||RBS-lysis1-DT||XbaI&PstI
 +
|-
 +
|3||RBS-lysis1-DT||XbaI&PstI
 +
|-
 +
|4||RBS-lysis1-DT||XbaI&PstI
 +
|}
 +
 +
[[File:igku_Aug19electrophoresis3]]<br>
 +
[[File:igku_Aug19electrophoresis4]]<br>
 +
{| class="wikitable"
 +
!Name||concentration[&micro;g/mL]||260/280||260/230
 +
|-
 +
|RBS-lacZ&alpha;-DT(EcoRI&SpeI)||9.8||1.50||0.39
 +
|-
 +
|Pcon-RBS-lacZ&alpha;-DT(EcoRI&SpeI)||11.2||1.47||0.08
 +
|-
 +
|}
 +
</div>
 +
 +
{| class="wikitable"
 +
!Lane||DNA||Enzyme
 +
|-
 +
|1||1kb ladder||--
 +
|-
 +
|3||RBS-lysis2-DT||XbaI&PstI
 +
|-
 +
|4||RBS-lysis2-DT||XbaI&PstI
 +
|-
 +
|5||RBS-lysis2-DT||XbaI&PstI
 +
|}
 +
 +
[[File:igku_Aug19electrophoresis3]]<br>
 +
[[File:igku_Aug19electrophoresis4]]<br>
 +
{| class="wikitable"
 +
!Name||concentration[&micro;g/mL]||260/280||260/230
 +
|-
 +
|RBS-lacZ&alpha;-DT(EcoRI&SpeI)||9.8||1.50||0.39
 +
|-
 +
|Pcon-RBS-lacZ&alpha;-DT(EcoRI&SpeI)||11.2||1.47||0.08
 +
|-
 +
|}
 +
</div>

Revision as of 13:03, 25 September 2013

Contents

Sep 13

Miniprep

Nakamoto

DNAconcentration[µg/mL]260/280260/230
9/12 Fusion6 antisense245.91.881.41
9/12 Plux-3115.51.581.32
9/12 Plux-4125.31.931.44
9/12 Fusion3in2 attenuator-1231.91.811.31

Gel Extraction

Tatsui,Ashida

LaneDNAEnzyme
11kb ladder--
2RBS-lysis1-DTXbaI&PstI
3RBS-lysis1-DTXbaI&PstI
4RBS-lysis1-DTXbaI&PstI

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Nameconcentration[µg/mL]260/280260/230
RBS-lacZα-DT(EcoRI&SpeI)9.81.500.39
Pcon-RBS-lacZα-DT(EcoRI&SpeI)11.21.470.08
LaneDNAEnzyme
11kb ladder--
3RBS-lysis2-DTXbaI&PstI
4RBS-lysis2-DTXbaI&PstI
5RBS-lysis2-DTXbaI&PstI

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Nameconcentration[µg/mL]260/280260/230
RBS-lacZα-DT(EcoRI&SpeI)9.81.500.39
Pcon-RBS-lacZα-DT(EcoRI&SpeI)11.21.470.08

</div>

LaneDNAEnzyme
3RBS-lysis3-DTXbaI&PstI
4RBS-lysis3-DTXbaI&PstI
5RBS-lysis3-DTXbaI&PstI
61kb ladder--

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Nameconcentration[µg/mL]260/280260/230
RBS-lacZα-DT(EcoRI&SpeI)9.81.500.39
Pcon-RBS-lacZα-DT(EcoRI&SpeI)11.21.470.08

</div>

LaneDNAEnzyme
21kb ladder--
3pSB1C3EcoRI&SpeI
4pSB1C3EcoRI&SpeI
5pSB1C3EcoRI&SpeI

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Nameconcentration[µg/mL]260/280260/230
RBS-lacZα-DT(EcoRI&SpeI)9.81.500.39
Pcon-RBS-lacZα-DT(EcoRI&SpeI)11.21.470.08

</div>

LaneDNAEnzyme
11kb ladder--
2pSB1C3XbaI&PstI
3pSB1C3XbaI&PstI
4pSB1C3XbaI&PstI

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Nameconcentration[µg/mL]260/280260/230
RBS-lacZα-DT(EcoRI&SpeI)9.81.500.39
Pcon-RBS-lacZα-DT(EcoRI&SpeI)11.21.470.08

</div>

Restriction Enzyme Digestion

Nakamoto

9/13 PlacEcoRISpeIXbaIPstI BufferBSAMilliQtotal
1cuts(PstI)160µL0µL0µL1µL3µL3µL7µL30µL
NC0.80µL0µL0µL0µL1µL1µL7.2µL10µL
9/10 Pcon attenuatorEcoRISpeIXbaIPstIBufferBSAMilliQtotal
1cut(PstI)100µL0µL0µL1µL3µL3µL13µL30µL
NC0.50µL0µL0µL0µL1µL1µL7.5µL10µL
9/12 Plux EcoRISpeIXbaIPstI BufferBSAMilliQtotal
1cuts(PstI)120µL0µL0µL1µL3µL3µL11µL30µL
NC0.60µL0µL0µL0µL1µL1µL7.4µL10µL
9/12 DT-EEcoRISpeIXbaIPstIBufferBSAMilliQtotal
1cut(XbaI)23.70µL0µL1µL0µL3µL3µL0µL30.7µL
NC1.20µL0µL0µL0µL1µL1µL6.8µL10µL
9/12 Pcon-RBS-luxR-bTE EcoRISpeIXbaIPstI BufferBSAMilliQtotal
1cuts(XbaI)22.40µL0µL1µL0µL3µL3µL0.6µL30µL
NC2.20µL0µL0µL0µL1µL1µL5.8µL10µL
9/12 Pcon-PEcoRISpeIXbaIPstIBufferBSAMilliQtotal
1cut(SpeI)210µL1µL0µL0µL3µL3µL12.0µL30µL
NC2.10µL0µL0µL0µL1µL1µL5.7µL10µL

M9 (EDTA)

Kanbayashi

1mM EDTA
volume10ml
Na2HPO4 300mg
KH2PO4150mg
NaCl25mg
NH4Cl50mg
Fe(3)-EDTA21mg　

5mM EDTA</div>

volume10ml
Na2HPO4300mg
KH2PO4150mg
NaCl25mg
NH4Cl50mg
Fe(3)-EDTA105mg　

</div>

Gel Extraction

Nakamoto

LaneDNAEnzyme
11kb ladder--
3Pcon-PSpeI
4Pcon-PSpeI
5Pcon-PSpeI

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Nameconcentration[µg/mL]260/280260/230
RBS-lacZα-DT(EcoRI&SpeI)9.81.500.39
Pcon-RBS-lacZα-DT(EcoRI&SpeI)11.21.470.08

Liquid Culture

Tatsui

Samplemedium
9/11 aptamer12_1M(pSB1C3)-1--
9/11 aptamer 12_P1--
9/11 Fusaion1 antisense-4--
9/12 pSB4K5-1--
9/12 pSB4K5-2--

Restriction Enzyme Digestion

Nakamoto

9/13 Plac-PEcoRISpeIXbaIPstI BufferBSAMilliQtotal
1cuts(SpeI)23.50µL0.5µL0µL0µL3µL3µL0µL30µL
NC80µL0µL0µL0µL1µL1µL0µL10µL
9/13 Pcon-pT181 attenuator-PEcoRISpeIXbaIPstIBufferBSAMilliQtotal
1cut(SpeI)23.50µL0.5µL0µL0µL3µL3µL0µL30µL
NC02.30µL0µL0µL0µL1µL1µL5.7µL10µL
9/13 Plux-P EcoRISpeIXbaIPstI BufferBSAMilliQtotal
1cuts(SpeI)23.80µL0.2µL0µL1µL3µL3µL0µL30µL
NC80µL0µL0µL0µL1µL1µL0µL10µL
9/5 RBS-lacZα-DTEcoRISpeIXbaIPstIBufferBSAMilliQtotal
2cut(XbaI&PstI)10.80µL0µL1µL1µL3µL3µL11.2µL30µL
NC0.50µL0µL0µL0µL1µL1µL7.5µL10µL

Electrophoresis

Hirano

LaneSample
1100bp ladder
2RBS-lacZα-DT(XbaI&PstI)
3RBS-lacZα-DT(NC9

File:Igku xxxxxxx.xxx

Ligation

No Name

stateVectorInserterLigation High ver.2
experiment9/13 Plac (SpeI&PstI)8.5µL9/13 pT181 antisense (XbaI&PstI)2.1 µL10.6 µL
experiment9/13 Plac (SpeI&PstI)8.5 µL9/10 aptamer 12_1R-DT (XbaI&PstI)1.8 µL10 .3µL
experiment9/13 Plac (SpeI&PstI)8.5 µL9/2 pT181 attenuator(XbaI&PstI)2.3 µL10.8 µL
experiment9/13 Plux (SpeI&PstI)11.4 µL9/13 RBS-lysis1-DT (XbaI&PstI)5.0 µL16.4 µL
experiment9/13 Plux (SpeI&PstI)11.4 µL9/13 RBS-lysis2-DT (XbaI&PstI)7.0 µL18.4 µL
experiment9/13 Plux (SpeI&PstI)7.2 µL9/13 RBS-lysis3-DT (XbaI&PstI)6.0 µL13.2 µL
experiment9/13 Pcon-RBS-luxR-DT (EcoRI&XbaI)2.0 µL9/4 Plux-RBS-GFP-DT (EcoRI&SpeI)5.2 µL72 µL
experiment9/13 Pcon-RBS-luxR-DT (EcoRI&XbaI)2.0 µL9/10 aptamer 12_1R-DT (XbaI&PstI)1.6 µL3.6 µL
experiment9/12 Ptet-pT181 antisense (--)1.0 µL9/10 spinach-DT (--)2.4 µL3.4 µL

PCR

No name

genome DNAKOD plus10x bufferdNTPMgSO4SasA_fwd primerSasA_rev primerMilliQtotal
0.52.52.51.50.750.75 25
PreDenatureDenatureAnnealingExtensioncycle
　°C　°C　°C　°C--
（◎Д◎）　　　　

PCR

No name

genome DNAKOD plus10x bufferdNTPMgSO4SasA_fwd primerSasA_rev primerMilliQtotal
0.52.52.51.50.750.75 25
PreDenatureDenatureAnnealingExtensioncycle
　°C　°C　°C　°C--
（◎Д◎）　　　　

Gel Extraction

Tatsui,Ashida

LaneDNAEnzyme
11kb ladder--
2RBS-lysis1-DTXbaI&PstI
3RBS-lysis1-DTXbaI&PstI
4RBS-lysis1-DTXbaI&PstI

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Nameconcentration[µg/mL]260/280260/230
RBS-lacZα-DT(EcoRI&SpeI)9.81.500.39
Pcon-RBS-lacZα-DT(EcoRI&SpeI)11.21.470.08
LaneDNAEnzyme
11kb ladder--
3RBS-lysis2-DTXbaI&PstI
4RBS-lysis2-DTXbaI&PstI
5RBS-lysis2-DTXbaI&PstI

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Nameconcentration[µg/mL]260/280260/230
RBS-lacZα-DT(EcoRI&SpeI)9.81.500.39
Pcon-RBS-lacZα-DT(EcoRI&SpeI)11.21.470.08
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