Template:Kyoto/Notebook/Sep 13

From 2013.igem.org

(Difference between revisions)
(→Gel Extraction)
(→PCR)
 
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Line 110: Line 110:
|1cut(PstI)||10||0µL||0µL||0µL||1µL||3µL||3µL||13µL||30µL
|1cut(PstI)||10||0µL||0µL||0µL||1µL||3µL||3µL||13µL||30µL
|-
|-
-
|NC||0.5||0µL||||0µL||0µL||0µL||1µL||1µL||7.5µL||10µL
+
|NC||0.5||0µL||0µL||0µL||0µL||1µL||1µL||7.5µL||10µL
|}
|}
Line 235: Line 235:
|1cut(SpeI)||23.5||0µL||0.5µL||0µL||0µL||3µL||3µL||0µL||30µL
|1cut(SpeI)||23.5||0µL||0.5µL||0µL||0µL||3µL||3µL||0µL||30µL
|-
|-
-
|NC||02.3||0µL||||0µL||0µL||0µL||1µL||1µL||5.7µL||10µL
+
|NC||02.3||0µL||0µL||0µL||0µL||1µL||1µL||5.7µL||10µL
|}
|}
Line 265: Line 265:
|2||RBS-lacZα-DT(XbaI&PstI)
|2||RBS-lacZα-DT(XbaI&PstI)
|-
|-
-
|3||RBS-lacZα-DT(NC9
+
|3||RBS-lacZα-DT(NC)
|}
|}
-
[[File:igku_xxxxxxx.xxx]]<br>
+
[[File:igku_91313.jpg]]<br>
</div>
</div>
Line 277: Line 277:
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
|-
|-
-
|experiment||9/13 Plac (SpeI&PstI)||8.5&micro;L||9/13 pT181 antisense (XbaI&PstI)||2.1 &micro;L||10.6 &micro;L
+
|experiment||9/13 Plac (SpeI&PstI)||8.5&micro;L||9/13 pT181 antisense (XbaI&PstI)||2.1 &micro;L||5.3 &micro;L
|-
|-
-
|experiment||9/13 Plac (SpeI&PstI)||8.5 &micro;L||9/10 aptamer 12_1R-DT (XbaI&PstI)||1.8 &micro;L||10 .3&micro;L
+
|experiment||9/13 Plac (SpeI&PstI)||8.5 &micro;L||9/10 aptamer 12_1R-DT (XbaI&PstI)||1.8 &micro;L||5.15&micro;L
|-
|-
-
|experiment||9/13 Plac (SpeI&PstI)||8.5 &micro;L||9/2 pT181 attenuator(XbaI&PstI)||2.3 &micro;L||10.8 &micro;L
+
|experiment||9/13 Plac (SpeI&PstI)||8.5 &micro;L||9/2 pT181 attenuator(XbaI&PstI)||2.3 &micro;L||10.4 &micro;L
|-
|-
-
|experiment||9/13 Plux (SpeI&PstI)||11.4 &micro;L||9/13 RBS-lysis1-DT (XbaI&PstI)||5.0 &micro;L||16.4 &micro;L
+
|experiment||9/13 Plux (SpeI&PstI)||11.4 &micro;L||9/13 RBS-lysis1-DT (XbaI&PstI)||5.0 &micro;L||8.2 &micro;L
|-
|-
-
|experiment||9/13 Plux (SpeI&PstI)||11.4 &micro;L||9/13 RBS-lysis2-DT (XbaI&PstI)||7.0 &micro;L||18.4 &micro;L
+
|experiment||9/13 Plux (SpeI&PstI)||11.4 &micro;L||9/13 RBS-lysis2-DT (XbaI&PstI)||7.0 &micro;L||9.2 &micro;L
|-
|-
-
|experiment||9/13 Plux (SpeI&PstI)||7.2 &micro;L||9/13 RBS-lysis3-DT (XbaI&PstI)||6.0 &micro;L||13.2 &micro;L
+
|experiment||9/13 Plux (SpeI&PstI)||7.2 &micro;L||9/13 RBS-lysis3-DT (XbaI&PstI)||6.0 &micro;L||6.6 &micro;L
|-
|-
-
|experiment||9/13 Pcon-RBS-luxR-DT (EcoRI&XbaI)||2.0 &micro;L||9/4 Plux-RBS-GFP-DT (EcoRI&SpeI)||5.2 &micro;L||72 &micro;L
+
|experiment||9/13 Pcon-RBS-luxR-DT (EcoRI&XbaI)||2.0 &micro;L||9/4 Plux-RBS-GFP-DT (EcoRI&SpeI)||5.2 &micro;L||3.6 &micro;L
|-
|-
-
|experiment||9/13 Pcon-RBS-luxR-DT (EcoRI&XbaI)||2.0 &micro;L||9/10 aptamer 12_1R-DT (XbaI&PstI)||1.6 &micro;L||3.6 &micro;L
+
|experiment||9/13 Pcon-RBS-luxR-DT (EcoRI&XbaI)||2.0 &micro;L||9/10 aptamer 12_1R-DT (XbaI&PstI)||1.6 &micro;L||1.8 &micro;L
|-
|-
-
|experiment||9/12 Ptet-pT181 antisense (--)||1.0 &micro;L||9/10 spinach-DT (--)||2.4 &micro;L||3.4 &micro;L
+
|experiment||9/12 Ptet-pT181 antisense (--)||1.0 &micro;L||9/10 spinach-DT (--)||2.4 &micro;L||1.7 &micro;L
|}
|}
Line 332: Line 332:
!genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total
!genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total
|-
|-
-
|8/20 J23100-RBS-lacZ&alpha;-DT||0.5||6.35||1.75||1.4||0.5||10.5
+
|8/20 J23100-RBS-lacZ&alpha;-DT||0.5||6.35||1.75||1.4||0.5||0||10.5
|}
|}
{| class="wikitable"
{| class="wikitable"
Line 347: Line 347:
!genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total
!genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total
|-
|-
-
|9/11 RBS-lysis1-DT||0.5||6.05||1.75||1.7||0.5||10.5
+
|9/11 RBS-lysis1-DT||0.5||6.05||1.75||1.7||0.5||0||10.5
|}
|}
{| class="wikitable"
{| class="wikitable"
Line 390: Line 390:
===PCR===
===PCR===
<div class="experiment">
<div class="experiment">
-
<span class="author">No name</span>
+
<span class="author">Yoshida</span>
{| class="wikitable"
{| class="wikitable"
-
!genome DNA||KOD plus||10x buffer||dNTP||MgSO4||SasA_fwd primer||SasA_rev primer||MilliQ||total
+
!genome DNA||KOD plus||10x buffer||dNTP||MgSO4||Prefix-RBS-RpaB_fwd primer||RpaB-GG4-suffix_rev primer||MilliQ||total
|-
|-
-
| ||0.5||2.5||2.5||1.5||0.75||0.75|| ||25
+
|RpaB||0.5||2.5||2.5||1.5||0.75||0.75||16.5||25
|}
|}
 +
{| class="wikitable"
 +
!genome DNA||KOD plus||10x buffer||dNTP||MgSO4||Prefix-PkaiBC_fwd primer ver.3||PkaiBC-suffix_rev primer ver.3||MilliQ||total
 +
|-
 +
|RpaB||0.5||2.5||2.5||1.5||0.75||0.75||16.5||25
 +
|}
 +
 +
{| class="wikitable"
{| class="wikitable"
!PreDenature||Denature||Annealing||Extension||cycle
!PreDenature||Denature||Annealing||Extension||cycle
Line 422: Line 429:
|}
|}
-
[[File:igku_Aug19electrophoresis3]]<br>
+
[[File:igku_91318.jpg]]<br>
-
[[File:igku_Aug19electrophoresis4]]<br>
+
[[File:igku_91319.jpg]]<br>
===Transformation===
===Transformation===

Latest revision as of 20:43, 27 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 Fusion3n2 attenuator-1231.91.811.31

Gel Extraction

Tatsui,Ashida

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

Igku 9131.jpg
Igku 9132.jpg

LaneDNAEnzyme
11kb ladder--
3RBS-lysis2-DTXbaI&PstI
4RBS-lysis2-DTXbaI&PstI
5RBS-lysis2-DTXbaI&PstI

Igku 9133.jpg
Igku 9134.jpg

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

Igku 9135.jpg
Igku 9136.jpg

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

Igku 9137.jpg
Igku 9138.jpg

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

Igku 9139.jpg
Igku 91310.jpg

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
volume10ml
Na2HPO4300mg
KH2PO4150mg
NaCl25mg
NH4Cl50mg
Fe(3)-EDTA105mg　

</div>

Gel Extraction

Nakamoto

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

Igku 91311.jpg
Igku 91312.jpg

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(NC)

Igku 91313.jpg

Ligation

No Name

stateVectorInserterLigation High ver.2
experiment9/13 Plac (SpeI&PstI)8.5µL9/13 pT181 antisense (XbaI&PstI)2.1 µL5.3 µL
experiment9/13 Plac (SpeI&PstI)8.5 µL9/10 aptamer 12_1R-DT (XbaI&PstI)1.8 µL5.15µL
experiment9/13 Plac (SpeI&PstI)8.5 µL9/2 pT181 attenuator(XbaI&PstI)2.3 µL10.4 µL
experiment9/13 Plux (SpeI&PstI)11.4 µL9/13 RBS-lysis1-DT (XbaI&PstI)5.0 µL8.2 µL
experiment9/13 Plux (SpeI&PstI)11.4 µL9/13 RBS-lysis2-DT (XbaI&PstI)7.0 µL9.2 µL
experiment9/13 Plux (SpeI&PstI)7.2 µL9/13 RBS-lysis3-DT (XbaI&PstI)6.0 µL6.6 µL
experiment9/13 Pcon-RBS-luxR-DT (EcoRI&XbaI)2.0 µL9/4 Plux-RBS-GFP-DT (EcoRI&SpeI)5.2 µL3.6 µL
experiment9/13 Pcon-RBS-luxR-DT (EcoRI&XbaI)2.0 µL9/10 aptamer 12_1R-DT (XbaI&PstI)1.6 µL1.8 µL
experiment9/12 Ptet-pT181 antisense (--)1.0 µL9/10 spinach-DT (--)2.4 µL1.7 µL

PCR

No name

genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/7 tetR aptamer 12_1R(pSB1C3) 0.56.051.751.70.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/6 pT181 antisense-1(pSB1C3) 0.56.051.751.70.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/6 pT181 attenuator (pSB1C3)0.53.851.753.90.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
8/27 Pλ-RBS-luxI-DT-10.52.551.755.20.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
8/31 Pbad/araC-RBS-RFP-20.53.751.7540.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/13 Plac(pSB1A2) 0.54.21.753.50.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
8/20 J23100-RBS-lacZα-DT0.56.351.751.40.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/7 Ptet-RBS-lacZα-DT0.56.051.751.70.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/8 J23100-spinach-DT0.55.751.7520.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/11 RBS-lysis1-DT0.56.051.751.70.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/12 RBS-lysis2-DT0.56.751.751.00.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/12 RBS-lysis3-DT0.56.751.751.00.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/8 J23100-spinach-DT0.56.351.751.400.510.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
8/27 Pλ-RBS-luxI-DT-10.52.551.755.200.510.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/12 RBS-lysis2-DT0.56.751.751.000.510.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/11 RBS-lysis1-DT0.56.051.751.700.510.5
PreDenatureDenatureAnnealingExtensioncycle
96°C96°C50°C68°C--
2m10s5s4m30

PCR

Yoshida

genome DNAKOD plus10x bufferdNTPMgSO4Prefix-RBS-RpaB_fwd primerRpaB-GG4-suffix_rev primerMilliQtotal
RpaB0.52.52.51.50.750.7516.525
genome DNAKOD plus10x bufferdNTPMgSO4Prefix-PkaiBC_fwd primer ver.3PkaiBC-suffix_rev primer ver.3MilliQtotal
RpaB0.52.52.51.50.750.7516.525


PreDenatureDenatureAnnealingExtensioncycle
94°C98°C58°C68°C--
2m10s30s30s30

Gel Extraction

Nakamoto

LaneDNAEnzyme
1100bp ladder--
3RBS-lacZα-DTXbaI&PstI
4RBS-lacZα-DTXbaI&PstI
5RBS-lacZα-DTXbaI&PstI
6RBS-lacZα-DTXbaI&PstI

Igku 91318.jpg
Igku 91319.jpg

Transformation

No name

NameSampleCompetent CellsTotalPlate
9/13 Plac+pT181 antisense2µL20µL22µLAmp
9/13 Plac+apt12_1R2µL20µL22µLAmp
9/13 Plac+pT181 attenuator2µL20µL22µLAmp
9/13 Plux+pT181 antisense2µL20µL22µLCP
9/13 Plux+RBS-lysis2-DT2µL20µL22µLCP
9/13 Plux+RBS-lysis2-DT2µL20µL22µLCP
9/13 Pcon-RBS-lacR-DT+Plux-RBS-GFP-DT2µL20µL22µLAmp
9/13 Pcon-pT181 antisense+apt12_1R-DT2µL20µL22µLAmp
9/13 Ptet-pT181antisense+spinach-DT2µL20µL22µLCP
Pλ-luxI-12µL20µL22µLAmp
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