Template:Kyoto/Notebook/Sep 2

From 2013.igem.org

(Difference between revisions)
(→Gel Extraction)
(→Sep 2)
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! ||pSB1C3-(1)||EcoRI||SpeI||XbaI||PstI||BufferB||BufferD||BSA||MilliQ||total
! ||pSB1C3-(1)||EcoRI||SpeI||XbaI||PstI||BufferB||BufferD||BSA||MilliQ||total
|-
|-
-
|2 cuts(E+S)||7µL||1µL||1µL||0µL||0µL||3µL||0µL||0.3µL||17.7µL||30µL
+
|2 cuts(EcoRI+SpeI)||7µL||1µL||1µL||0µL||0µL||3µL||0µL||0.3µL||17.7µL||30µL
|-
|-
-
|NC(E+S)||0.3µL||0µL||0µL||0µL||0µL||1µL||0µL||0.1µL||8.6µL||10µL
+
|NC(EcoRI+SpeI)||0.3µL||0µL||0µL||0µL||0µL||1µL||0µL||0.1µL||8.6µL||10µL
|-
|-
-
|2 cuts(X+P)||7µL||0µL||0µL||1µL||1µL||0µL||3µL||0.3µL||17.7µL||30µL
+
|2 cuts(XbaI+PstI)||7µL||0µL||0µL||1µL||1µL||0µL||3µL||0.3µL||17.7µL||30µL
|-
|-
-
|NC(X+P)||0.3µL||0µL||0µL||0µL||0µL||0µL||1µL||0.1µL||8.6µL||10µL
+
|NC(XbaI+PstI)||0.3µL||0µL||0µL||0µL||0µL||0µL||1µL||0.1µL||8.6µL||10µL
|}
|}
{| class="wikitable"
{| class="wikitable"
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! ||8/21 pT181 attenuator-(2)||EcoRI||SpeI||XbaI||PstI||BufferB||BufferD||BSA||MilliQ||total
! ||8/21 pT181 attenuator-(2)||EcoRI||SpeI||XbaI||PstI||BufferB||BufferD||BSA||MilliQ||total
|-
|-
-
|2 cuts(E+S)||8µL||1µL||1µL||0µL||0µL||3µL||0µL||0.3µL||16.3µL||30µL
+
|2 cuts(EcoRI+SpeI)||8µL||1µL||1µL||0µL||0µL||3µL||0µL||0.3µL||16.3µL||30µL
|-
|-
-
|NC(E+S)||0.5µL||0µL||0µL||0µL||0µL||1µL||0µL||0.1µL||8.4µL||10µL
+
|NC(EcoRI+SpeI)||0.5µL||0µL||0µL||0µL||0µL||1µL||0µL||0.1µL||8.4µL||10µL
|-
|-
-
|2 cut(X+P)s||8µL||0µL||0µL||1µL||1µL||0µL||3µL||0.3µL||16.3µL||30µL
+
|2 cuts(XbaI+PstI)||8µL||0µL||0µL||1µL||1µL||0µL||3µL||0.3µL||16.3µL||30µL
|-
|-
-
|NC(X+P)||0.5µL||0µL||0µL||0µL||0µL||0µL||1µL||0.1µL||8.4µL||10µL
+
|NC(XbaI+PstI)||0.5µL||0µL||0µL||0µL||0µL||0µL||1µL||0.1µL||8.4µL||10µL
|}
|}
[[File:Igku Sep2 Restriction Enzyme Digestion (N1-1).jpg]]<br>
[[File:Igku Sep2 Restriction Enzyme Digestion (N1-1).jpg]]<br>
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|1||100bp ladder||-
|1||100bp ladder||-
|-
|-
-
|2||rowspan="2"|pSB1C3(E+S)||rowspan="2"|EcoRI&SpeI
+
|2||rowspan="2"|pSB1C3(EcoRI+SpeI)||rowspan="2"|EcoRI&SpeI
|-
|-
|3
|3
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|1||100bp ladder||-
|1||100bp ladder||-
|-
|-
-
|2||rowspan="2"|pT181 attenuator(2)(E+S)||rowspan="2"|EcoRI&SpeI
+
|2||rowspan="2"|pT181 attenuator(2)(EcoRI+SpeI)||rowspan="2"|EcoRI&SpeI
|-
|-
|3
|3
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|4||--||--
|4||--||--
|-
|-
-
|5||rowspan="2"|pT181 attenuator(2)(X+P)||rowspan="2"|EcoRI&SpeI
+
|5||rowspan="2"|pT181 attenuator(2)(XbaI+PstI)||rowspan="2"|EcoRI&SpeI
|-
|-
|6
|6
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|7||--||--
|7||--||--
|-
|-
-
|8||rowspan="2"|pT181 attenuator(2)(X+P)||rowspan="2"|EcoRI&SpeI
+
|8||rowspan="2"|pT181 attenuator(2)(XbaI+PstI)||rowspan="2"|EcoRI&SpeI
|-
|-
|9
|9

Revision as of 05:20, 25 September 2013

Contents

Sep 2

Colony PCR

Tatsui

Samplebase pair
9/1 RBS-lysis2-DT-(1)985
Ptet-RBS-lacZα-DT-(1)765
Plac-RBS-lacZα-DT-(1)765
Plac-RBS-lacZα-DT-(2)765
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min30cycles

Igku Sep2 ColonyPCR (N1-2).jpg

Restriction Enzyme Digestion

Nakamoto

pSB1C3-(1)EcoRISpeIXbaIPstIBufferBBufferDBSAMilliQtotal
2 cuts(EcoRI+SpeI)7µL1µL1µL0µL0µL3µL0µL0.3µL17.7µL30µL
NC(EcoRI+SpeI)0.3µL0µL0µL0µL0µL1µL0µL0.1µL8.6µL10µL
2 cuts(XbaI+PstI)7µL0µL0µL1µL1µL0µL3µL0.3µL17.7µL30µL
NC(XbaI+PstI)0.3µL0µL0µL0µL0µL0µL1µL0.1µL8.6µL10µL
8/21 tRMA-spinach(1)EcoRISpeIBufferBBSAMilliQtotal
2 cuts8µL1.0µL1.0µL3µL0.3µL17.6µL30µL
NC0.5µL0µL0µL1µL0.1µL8.4µL10µL
8/21 tetR aptamer12_1R-(1)EcoRISpeIBufferBBSAMilliQtotal
2 cuts6µL1.0µL1.0µL3µL0.3µL18.7µL30µL
NC0.4µL0µL0µL1µL0.1µL8.5µL10µL
8/21 pT181 attenuator-(2)EcoRISpeIXbaIPstIBufferBBufferDBSAMilliQtotal
2 cuts(EcoRI+SpeI)8µL1µL1µL0µL0µL3µL0µL0.3µL16.3µL30µL
NC(EcoRI+SpeI)0.5µL0µL0µL0µL0µL1µL0µL0.1µL8.4µL10µL
2 cuts(XbaI+PstI)8µL0µL0µL1µL1µL0µL3µL0.3µL16.3µL30µL
NC(XbaI+PstI)0.5µL0µL0µL0µL0µL0µL1µL0.1µL8.4µL10µL

Igku Sep2 Restriction Enzyme Digestion (N1-1).jpg

Liquid Culture

Kojima

Samplemedium
tRNA-Spinach-1Plusgrow medium(+CP)
tetR aptamaer12_1R-1Plusgrow medium(+CP)
pT181 attenuator-1Plusgrow medium(+CP)　
pT181 antisense-1Plusgrow medium(+CP)

Master Plate

Kojima

NumberUse LB plate(+CP)
1tRNA Spinach-1
2tetR aptamaer12_1R
3tetR aptamaer12_P
4tetR aptamaer12_1M
5pT181 attenuator-2
6Fusion1 attenuator-1
7Fusion3m2 attenuator-1
8pT181 antisense-1
9Fusion1 antisense-1
10Fuaion6 antisense-1

Electrophoresis

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder-
2pSB1C3(EcoRI+SpeI)EcoRI&SpeI
3
4----
58/24 tetR aptamer 12_1REcoRI&SpeI
6

Igku Sep1 Gel Extraction(N2-1).jpg
Igku Sep1 Gel Extraction(N2-2).jpg
No name

LaneDNAEnzyme
1100bp ladder-
2pT181 attenuator(2)(EcoRI+SpeI)EcoRI&SpeI
3
4----
5pT181 attenuator(2)(XbaI+PstI)EcoRI&SpeI
6
7----
8pT181 attenuator(2)(XbaI+PstI)EcoRI&SpeI
9
10----
11SpinachEcoRI&SpaI
12

Igku Sep1 Gel Extraction(N3-1).jpg
Igku Sep1 Gel Extraction(N3-2).jpg

Nameconcentration[µg/mL]260/280260/230
pSB1C3 (EcoRI&SpeI)3.02.180.31
pSB1C3 (XbaI&PstI)4.72.250.36
pT181 attenuator-(2)(EcoR&SpeI)8.62.740.01
pT181 attenuator-(2) (XbaI&PstI)16.52.460.03
Spinach (EcoRI&SpeI)2.82.980.27
tetR aptamer12_1R (EcoRI&SpeI)50.428.071.97

Colony PCR

No name

Samplebase pair
9/1 Spinach(pSB1C3)about 450
9/1 Spinach-DT596
9/1 RBS-lysis1-DT613
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s40s30cycles

Igku Sep2 ColonyPCR (N4).jpg

Samplebase pair
9/1 Ptet-RBS-lacZα-DT-2738
9/1 Ptet-RBS-lacZα-DT-3738
9/1 Ptet-RBS-lacZα-DT-4738
9/1 Ptet-RBS-lacZα-DT-5738
9/1 Ptet-RBS-lacZα-DT-6738
9/1 RBS-lysis2-DT-2985
9/1 RBS-lysis2-DT-3985
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min30cycles

Igku Sep3 ColonyPCR (N1).jpg

Master Plate

No name

NumberUse LB plate(+CP)
19/1 Spinach(pSB1C3)
29/1 Spinach-DT-(1)

Liquid Culture

No name

Samplemedium
pT181 attenuatorPlusgrow medium (+CP)
pT181 antisensePlusgrow medium (+CP)
SpinachPlusgrow medium (+CP)
  • incubated at 37 °C for 1 hour

Transformation

No name

NameSampleCompetent CellsTotalPlate
8/28 RBS-lysis3-DT3µL30µL33µLCP
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