Template:Kyoto/Notebook/Sep 12

From 2013.igem.org

(Difference between revisions)
(→Gel Extraction)
(→Sep 12)
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</div>
</div>
 +
<div class="experiment">
 +
<span class="author">Honda</span>
 +
{|===Colony PCR===
 +
class="wikitable"
 +
!Sample||base pair
 +
|-
 +
|9/11 apt12-1M(pSB1C3) 1||513
 +
|-
 +
|9/11 apt12-1M(pSB1C3) 2||513
 +
|-
 +
|9/11 Fusion3m2a09ttenuator(pSB1C3) 1||609
 +
|--
 +
|9/11 Fusion6 antisense(pSB1C3)1||431-
 +
|-
 +
|9/11 aptamer 12-P(pSB1C3)1||515
 +
|-
 +
|9/11 Fusion1 antisense(pSB1C3) 1||420
 +
|-
 +
|9/11 Fusion1 antisense(pSB1C3) 2||420
 +
|-
 +
|9/11 Plac(BBa-R0011) 1||293
 +
|-
 +
|9/11 Plac(BBa-R0011) 2||293
 +
|-
 +
|9/11 Plac(BBa-R0011) 3||293
 +
|-
 +
|9/11 Plac(BBa-R0011) 4||293
 +
|-
 +
|9/11 Pcon-luxR-Plux-GFP 1||2138
 +
|-
 +
|9/11 Pcon-RBS-lacZ&alpha;-DT(pSB4K5) 1||712
 +
|
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||--
 +
|-
 +
|5min||30s||30s||2min12s||30cycles
 +
|}
 +
</div>
 +
 +
===Restriction Enzyme Digestion===
 +
<div class="experiment">
 +
<span class="author">Tatsui</span>
 +
{| class="wikitable"
 +
! ||9/5 Pcon ||EcoRI||SpeI||XbaI||PstI|| Buffer||BSA||MilliQ||total
 +
|-
 +
|1cuts(P)||0&micro;L||0&micro;L||0&micro;L||1&micro;L||3&micro;L||3&micro;L||17.3&micro;L||30&micro;L
 +
|-
 +
|NC(P)||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||6.1&micro;L||10&micro;L
 +
|-
 +
|}
 +
{| class="wikitable"
 +
! ||8/17 DT||EcoR1||Buffer||BSA||MilliQ||total
 +
|-
 +
|1cut||1&micro;L||3&micro;L||3&micro;L||12.4&micro;L||30&micro;L|
 +
|-
 +
|NC||0&micro;L||1&micro;L||1&micro;L||4.5&micro;L||10&micro;L|
 +
|}
 +
===Colony PCR===
 +
<div class="experiment">
 +
<span class="author">Nakamoto,Tatsui</span>
 +
{| class="wikitable"
 +
!Sample||base pair
 +
|-
 +
|9/11 Pcon-pT181atte-RBS-lacZ&alpha;-DT||965
 +
|-
 +
|9/11 Plux-PBS-lysis1-DT 1||613
 +
|-
 +
|9/11 Pcon-pT181attenuator-aptamer12-1R-DT 1||859
 +
|-
 +
|9/11 Pcon-pT181attenuator-aptamer12-1R-DT 2||859
 +
|-
 +
|9/11 Plux\RBS-lysis3-DT 1||1210
 +
|-
 +
|9/11 Plux\RBS-lysis3-DT 2||1210
 +
|-
 +
|Pcon-Spinach-DT(pSB4K5) 1||605
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||--
 +
|-
 +
|5min||30s||30s||1min12s||30cycles
 +
|}
 +
</div>
 +
===Transformation===
 +
<div class="experiment">
 +
<span class="author">tatsui</span>
 +
{| class="wikitable"
 +
!Name||Sample||Competent Cells||Plate
 +
|-
 +
|}
 +
</div>
 +
 +
{| class="wikitable"
 +
!Lane||Sample
 +
|-
 +
|1||1kb ladder
 +
|-
 +
|2||P&lambda-RBS-luxI-DT(PCR product)
 +
|-
 +
|3||Pbad/araC-RBS-RFP(PCR product)
 +
|}
 +
 +
[[File:igku_xxxxxx.xxx]]
 +
 +
</div>
 +
 +
===Electrophoresis===
 +
<div class="experiment">
 +
<span class="author">No name </span></div>
 +
1
 +
{| class="wikitable"
 +
!Lane||Sample
 +
|-
 +
|1||Fusion6 antisense
 +
|-
 +
|2||Fusion1 antisense-1
 +
|-
 +
|3|| Fusion1 antisense-2
 +
|-
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|4||100bp ladder
 +
|-
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|5||Plac(BBa-R0011) 1
 +
|-
 +
|6|| Plac(BBa-R0011) 2
 +
|-
 +
|7|| Plac(BBa-R0011) 3
 +
|-
 +
|8|| Plac(BBa-R0011) 4
 +
|}
 +
 +
[[File:igku_xxxxxx.xxx]]
 +
 +
</div>
 +
 +
2
 +
{| class="wikitable"
 +
!Lane||Sample
 +
|-
 +
|1||1kb ladder
 +
|-
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|2||aptamer12-1M 1
 +
|-
 +
|3|| aptamer12-1M 2
 +
|-
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|4||Fusion3m2 attenuator
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|-
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|5||aptamer12-P1
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|-
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|6||Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT
 +
|-
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|7||Pcon-RBS-lacZ&alpha;=DT
 +
|-
 +
|8|| 1kb ladder
 +
|}

Revision as of 16:19, 23 September 2013

Contents

Sep 12

Miniprep

Nakamoto

DNAconcentration[µg/mL]260/280260/230
Pcon-pT181atte-DT293.6　1.88　1.84
Pcon-apt12-1R-DT303.8　1.75　1.64
Fusion1atte(pSB1c3)172.0　1.96　2.31
RBS-lisis1-DT235.11.591.60
RBS-lisis2-DT387.71.912.01
RBS-lisis3-DT387.91.912.09
Plux166.71.981.63

Electrophoresis

Nakamoto

LaneSample
1100bp ladder
29/11 P-RBS-lisis1-DT(Colony PCR prodution)

File:Igku xxxxxx.xxx

Liquid Culture

Nakamoto

Samplemedium
9/10 Pcon-apt12-1R-DT1Plusgrow medium(+Amp)
9/10 DT-Pcon-pT181atte1Plusgrow medium(+CP)
9/10 Fusion1-atte(pSB1c3)Plusgrow medium(+CP)　
v>

Electrophoresis

Tatsui

1

LaneSample
11kb ladder
2pSB4K5(EcoR1&Spe1)
3 pSB4K5 NC(EcoR1&Spe1)
4RBS-lacz-DT(EcoR1&Spe1)
5 EcoR1&Spe1 NC( EcoR1&Spe1)
6Pcon-RBS-lacz-DT( EcoR1&Spe1)
7 Pcon-RBS-lacz-DT NC( EcoR1&Spe1)
81kb ladder

File:Igku xxxxxx.xxx

2

LaneSample
11kb ladder
2Ptet-pT181anti(Spe1&Pst1)
3 Ptet-pT181anti NC(Spe1&Pst1)
4RBS-lysis2-DT(Xba1&Pst1)
5 EcoR1&Spe1 NC( EcoR1&Spe1)
6--
7--
81kb ladder

File:Igku xxxxxx.xxx

Gel Extraction

Tatsui

LaneDNAEnzyme
11kb ladder--
2RBS-lacZ-DTEcoR1&Spe1
3RBS-lacZ-DTEcoR1&Spe1
4RBS-lacZ-DTEcoR1&Spe1
5----
6Pcon-RBS-lacZ-DTEcoR1&Spe1
7Pcon-RBS-lacZ-DTEcoR1&Spe1
8Pcon-RBS-lacZ-DTEcoR1&Spe1
91kb ladder--

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Nameconcentration[µg/mL]260/280260/230
RBS-lacZ-DT(EcoR1&Spe1)9.81.500.39
Pcon-RBS-lacZ-DT(EcoR1&Spe1)11.21.470.08

Gel Extraction

Tatsui

LaneDNAEnzyme
11kb ladder--
2RBS-lysis2-DTXba1&Pst1
3RBS-lysis2-DTXba1&Pst1
4RBS-lysis2-DTXba1&Pst1
5----
61kb ladder--

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Nameconcentration[µg/mL]260/280260/230
RBS-lysis2-DT(Xba1&Pst1)5.41.430.77

Honda

class="wikitable"
Samplebase pair
9/11 apt12-1M(pSB1C3) 1513
9/11 apt12-1M(pSB1C3) 2513
9/11 Fusion3m2a09ttenuator(pSB1C3) 1609
9/11 Fusion6 antisense(pSB1C3)1431-
9/11 aptamer 12-P(pSB1C3)1515
9/11 Fusion1 antisense(pSB1C3) 1420
9/11 Fusion1 antisense(pSB1C3) 2420
9/11 Plac(BBa-R0011) 1293
9/11 Plac(BBa-R0011) 2293
9/11 Plac(BBa-R0011) 3293
9/11 Plac(BBa-R0011) 4293
9/11 Pcon-luxR-Plux-GFP 12138
9/11 Pcon-RBS-lacZα-DT(pSB4K5) 1712
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s2min12s30cycles

Restriction Enzyme Digestion

Tatsui

9/5 Pcon EcoRISpeIXbaIPstI BufferBSAMilliQtotal
1cuts(P)0µL0µL0µL1µL3µL3µL17.3µL30µL
NC(P)0µL0µL0µL0µL1µL1µL6.1µL10µL
8/17 DTEcoR1BufferBSAMilliQtotal
1cut1µL3µL3µL12.4µL
NC0µL1µL1µL4.5µL

Colony PCR

Nakamoto,Tatsui

Samplebase pair
9/11 Pcon-pT181atte-RBS-lacZα-DT965
9/11 Plux-PBS-lysis1-DT 1613
9/11 Pcon-pT181attenuator-aptamer12-1R-DT 1859
9/11 Pcon-pT181attenuator-aptamer12-1R-DT 2859
9/11 Plux\RBS-lysis3-DT 11210
9/11 Plux\RBS-lysis3-DT 21210
Pcon-Spinach-DT(pSB4K5) 1605
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min12s30cycles

Transformation

tatsui

NameSampleCompetent CellsPlate
LaneSample
11kb ladder
2P&lambda-RBS-luxI-DT(PCR product)
3Pbad/araC-RBS-RFP(PCR product)

File:Igku xxxxxx.xxx

Electrophoresis

No name

1

LaneSample
1Fusion6 antisense
2Fusion1 antisense-1
3 Fusion1 antisense-2
4100bp ladder
5Plac(BBa-R0011) 1
6 Plac(BBa-R0011) 2
7 Plac(BBa-R0011) 3
8 Plac(BBa-R0011) 4

File:Igku xxxxxx.xxx

</div>

2

LaneSample
11kb ladder
2aptamer12-1M 1
3 aptamer12-1M 2
4Fusion3m2 attenuator
5aptamer12-P1
6Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT
7Pcon-RBS-lacZα=DT
8 1kb ladder
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