Template:Kyoto/Notebook/Sep 2

From 2013.igem.org

(Difference between revisions)
(→Colony PCR)
(→Restriction Enzyme Digestion)
 
(21 intermediate revisions not shown)
Line 1: Line 1:
==Sep 2==
==Sep 2==
-
 
-
===Liquid Culture===
 
-
<div class="experiment">
 
-
<span class="author">Hirano</span>
 
-
{| class="wikitable"
 
-
!Sample||medium
 
-
|-
 
-
|9/1 entA-(Master Plate)-1||4mL SOB(+Km)
 
-
|}
 
-
 
-
37&deg;C
 
===Colony PCR===
===Colony PCR===
Line 20: Line 9:
|9/1 RBS-lysis2-DT-(1)||985
|9/1 RBS-lysis2-DT-(1)||985
|-
|-
-
|Ptet-RBS-lacZα-DT-(1)||765
+
|Ptet-RBS-lacZ&alpha;-DT-(1)||765
|-
|-
-
|Plac-RBS-lacZα-DT-(1)||765
+
|Plac-RBS-lacZ&alpha;-DT-(1)||765
|-
|-
-
|Plac-RBS-lacZα-DT-(2)||765
+
|Plac-RBS-lacZ&alpha;-DT-(2)||765
|}
|}
{| class="wikitable"
{| class="wikitable"
Line 33: Line 22:
|5min||30s||30s||1min||30cycles
|5min||30s||30s||1min||30cycles
|}
|}
-
[[File:Igku Sep2 ColonyPCR (N1-2).jpg]]
+
</div>
 +
===Electrophoresis===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Lane||Sample
 +
|-
 +
|1||100bp ladder
 +
|-
 +
|2||9/1 RBS-lysis2+DT -(1)
 +
|-
 +
|3||9/1 Ptet+RBS-lacZ&alpha;-DT -(1)
 +
|-
 +
|4||9/1 Plac+RBS-lacZ&alpha;-DT -(1)
 +
|-
 +
|5||9/1 Plac+RBS-lacZ&alpha;-DT -(2)
 +
|-
 +
|6||100bp ladder
 +
|}
 +
[[File:Igku Sep2 ColonyPCR (N1-2).jpg]]<br>
</div>
</div>
Line 42: Line 50:
! ||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)||7&micro;L||1&micro;L||1&micro;L||0&micro;L||0&micro;L||3&micro;L||0&micro;L||0.3&micro;L||17.7&micro;L||30&micro;L
+
|2 cuts(EcoRI+SpeI)||7&micro;L||1&micro;L||1&micro;L||0&micro;L||0&micro;L||3&micro;L||0&micro;L||0.3&micro;L||17.7&micro;L||30&micro;L
|-
|-
-
|NC(E+S)||0.3&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||0&micro;L||0.1&micro;L||8.6&micro;L||10&micro;L
+
|NC(EcoRI+SpeI)||0.3&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||0&micro;L||0.1&micro;L||8.6&micro;L||10&micro;L
|-
|-
-
|2 cut(X+P)s||7&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||0&micro;L||3&micro;L||0.3&micro;L||17.7&micro;L||30&micro;L
+
|2 cuts(XbaI+PstI)||7&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||0&micro;L||3&micro;L||0.3&micro;L||17.7&micro;L||30&micro;L
|-
|-
-
|NC(X+P)||0.3&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||8.6&micro;L||10&micro;L
+
|NC(XbaI+PstI)||0.3&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||8.6&micro;L||10&micro;L
|}
|}
{| class="wikitable"
{| class="wikitable"
Line 67: Line 75:
! ||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)||8&micro;L||1&micro;L||1&micro;L||0&micro;L||0&micro;L||3&micro;L||0&micro;L||0.3&micro;L||16.3&micro;L||30&micro;L
+
|2 cuts(EcoRI+SpeI)||8&micro;L||1&micro;L||1&micro;L||0&micro;L||0&micro;L||3&micro;L||0&micro;L||0.3&micro;L||16.3&micro;L||30&micro;L
|-
|-
-
|NC(E+S)||0.5&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||0&micro;L||0.1&micro;L||8.4&micro;L||10&micro;L
+
|NC(EcoRI+SpeI)||0.5&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||0&micro;L||0.1&micro;L||8.4&micro;L||10&micro;L
|-
|-
-
|2 cut(X+P)s||8&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||0&micro;L||3&micro;L||0.3&micro;L||16.3&micro;L||30&micro;L
+
|2 cuts(XbaI+PstI)||8&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||0&micro;L||3&micro;L||0.3&micro;L||16.3&micro;L||30&micro;L
|-
|-
-
|NC(X+P)||0.5&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||8.4&micro;L||10&micro;L
+
|NC(XbaI+PstI)||0.5&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||8.4&micro;L||10&micro;L
|}
|}
[[File:Igku Sep2 Restriction Enzyme Digestion (N1-1).jpg]]<br>
[[File:Igku Sep2 Restriction Enzyme Digestion (N1-1).jpg]]<br>
Line 89: Line 97:
|-
|-
|pT181 attenuator-1||Plusgrow medium(+CP)　
|pT181 attenuator-1||Plusgrow medium(+CP)　
-
|=
+
|-
|pT181 antisense-1||Plusgrow medium(+CP)
|pT181 antisense-1||Plusgrow medium(+CP)
|}
|}
Line 120: Line 128:
|10||Fuaion6 antisense-1
|10||Fuaion6 antisense-1
|}  
|}  
 +
</div>
 +
 +
===Electrophoresis===
 +
<div class="experiment">
 +
<span class="author">Nakamoto & Tatsui</span>
 +
{| class="wikitable"
 +
!Lane||Sample||Enzyme1||Enzyme2
 +
|-
 +
|1||100bp ladder||--||--
 +
|-
 +
|2||pSB1C3 -(1)||EcoRI||SpeI
 +
|-
 +
|3||pSB1C3 -(1)||--||--
 +
|-
 +
|5||pSB1C3 -(1)||XbaI||PstI
 +
|-
 +
|6||pSB1C3 -(1)||--||--
 +
|-
 +
|7||pT181 attenuator (2)||EcoRI||SpeI
 +
|-
 +
|8||pT181 attenuator (2)||--||--
 +
|-
 +
|9||pT181 attenuator (2)||XbaI||SpeI
 +
|-
 +
|10||pT181 attenuator (2)||--||--
 +
|-
 +
|11||tetR-aptamer 12_1R||EcoRI||SpeI
 +
|-
 +
|12||tetR-aptamer 12_1R||--||--
 +
|-
 +
|13||tRNA Spinach (1)||EcoRI||SpeI
 +
|-
 +
|14||100bp ladder||--||--
 +
|-
 +
|15||tRNA Spinach (1)||--||--
 +
|}
 +
[[File:Igku Sep2 Restriction Enzyme Digestion (N1-1).jpg]]<br>
</div>
</div>
Line 130: Line 175:
|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
Line 142: Line 187:
[[File:Igku Sep1 Gel Extraction(N2-1).jpg]]<br>
[[File:Igku Sep1 Gel Extraction(N2-1).jpg]]<br>
[[File:Igku Sep1 Gel Extraction(N2-2).jpg]]<br>
[[File:Igku Sep1 Gel Extraction(N2-2).jpg]]<br>
-
<span class="author">No name</span>
 
{| class="wikitable"
{| class="wikitable"
!Lane||DNA||Enzyme
!Lane||DNA||Enzyme
Line 148: Line 192:
|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
Line 154: Line 198:
|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
Line 160: Line 204:
|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
Line 208: Line 252:
|5min||30s||30s||40s||30cycles
|5min||30s||30s||40s||30cycles
|}
|}
-
[[File:Igku Sep2 ColonyPCR (N4).jpg]]
 
{| class="wikitable"
{| class="wikitable"
!|Sample||base pair
!|Sample||base pair
|-
|-
-
|9/1 Ptet-RBS-lacZα-DT-2||738
+
|9/1 Ptet-RBS-lacZ&alpha;-DT-2||738
|-
|-
-
|9/1 Ptet-RBS-lacZα-DT-3||738
+
|9/1 Ptet-RBS-lacZ&alpha;-DT-3||738
|-
|-
-
|9/1 Ptet-RBS-lacZα-DT-4||738
+
|9/1 Ptet-RBS-lacZ&alpha;-DT-4||738
|-
|-
-
|9/1 Ptet-RBS-lacZα-DT-5||738
+
|9/1 Ptet-RBS-lacZ&alpha;-DT-5||738
|-
|-
-
|9/1 Ptet-RBS-lacZα-DT-6||738
+
|9/1 Ptet-RBS-lacZ&alpha;-DT-6||738
|-
|-
|9/1 RBS-lysis2-DT-2||985
|9/1 RBS-lysis2-DT-2||985
Line 234: Line 277:
|}
|}
[[File:Igku Sep3 ColonyPCR (N1).jpg]]
[[File:Igku Sep3 ColonyPCR (N1).jpg]]
 +
</div>
 +
 +
===Electrophoresis===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Lane||Sample
 +
|-
 +
|1||100bp ladder
 +
|-
 +
|2||Spinach(pSB1C3)
 +
|-
 +
|3||Spinach-DT
 +
|-
 +
|4||RBS-lysis1-DT
 +
|-
 +
|5||100bp ladder
 +
|}
 +
[[File:Igku Sep2 ColonyPCR (N4).jpg]]<br>
</div>
</div>
Line 246: Line 308:
|2||9/1 Spinach-DT-(1)
|2||9/1 Spinach-DT-(1)
|}
|}
-
<!-- 残りはハズレ -->
 
</div>
</div>
Line 261: Line 322:
|Spinach||Plusgrow medium (+CP)
|Spinach||Plusgrow medium (+CP)
|}
|}
-
* 37&dig:C ihour incubate
+
*incubated at 37 &deg;C for 1 hour
</div>
</div>

Latest revision as of 18:50, 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

Electrophoresis

No name

LaneSample
1100bp ladder
29/1 RBS-lysis2+DT -(1)
39/1 Ptet+RBS-lacZα-DT -(1)
49/1 Plac+RBS-lacZα-DT -(1)
59/1 Plac+RBS-lacZα-DT -(2)
6100bp ladder

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

Nakamoto & Tatsui

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2pSB1C3 -(1)EcoRISpeI
3pSB1C3 -(1)----
5pSB1C3 -(1)XbaIPstI
6pSB1C3 -(1)----
7pT181 attenuator (2)EcoRISpeI
8pT181 attenuator (2)----
9pT181 attenuator (2)XbaISpeI
10pT181 attenuator (2)----
11tetR-aptamer 12_1REcoRISpeI
12tetR-aptamer 12_1R----
13tRNA Spinach (1)EcoRISpeI
14100bp ladder----
15tRNA Spinach (1)----

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

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

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
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

Electrophoresis

No name

LaneSample
1100bp ladder
2Spinach(pSB1C3)
3Spinach-DT
4RBS-lysis1-DT
5100bp ladder

Igku Sep2 ColonyPCR (N4).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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