Template:Kyoto/Notebook/Sep 2

From 2013.igem.org

(Difference between revisions)
(→Sep 2)
(→Colony PCR)
Line 194: Line 194:
!|Sample||base pair
!|Sample||base pair
|-
|-
-
|8/27 pT181 attenuator-DT-1||738
+
|9/1 Spinach(pSB1C3)||about 450
|-
|-
-
|8/27 pT181 attenuator-DT-2||738
+
|9/1 Spinach-DT||596
|-
|-
-
|8/27 pT181 attenuator-DT-3||738
+
|9/1 RBS-lysis1-DT||613
-
|-
+
-
|8/27 TetR-aptamer 12_1R-DT-1||521
+
-
|-
+
-
|8/27 TetR-aptamer 12_1R-DT-2||521
+
-
|-
+
-
|8/27 pT181 antisense-DT-1||542
+
|}
|}
{| class="wikitable"
{| class="wikitable"
Line 211: Line 205:
|94°C||94°C||55°C||68°C||--
|94°C||94°C||55°C||68°C||--
|-
|-
-
|5min||30s||30s||30s||30cycles
+
|5min||30s||30s||40s||30cycles
|}
|}
-
[[File:igku_Aug28electrophoresis3?.png]]
+
[[File:igku_sep2electrophoresis1.png]]
{| class="wikitable"
{| class="wikitable"
!|Sample||base pair
!|Sample||base pair
|-
|-
-
|8/27 Pλ-luxI-1||???
+
|9/1 Ptet-RBS-lacZα-DT-2||738
 +
|-
 +
|9/1 Ptet-RBS-lacZα-DT-3||738
 +
|-
 +
|9/1 Ptet-RBS-lacZα-DT-4||738
 +
|-
 +
|9/1 Ptet-RBS-lacZα-DT-5||738
 +
|-
 +
|9/1 Ptet-RBS-lacZα-DT-6||738
 +
|-
 +
|9/1 RBS-lysis2-DT-2||985
|-
|-
-
|8/27 Pλ-luxI||???
+
|9/1 RBS-lysis2-DT-3||985
|}
|}
{| class="wikitable"
{| class="wikitable"
Line 226: Line 230:
|94°C||94°C||55°C||68°C||--
|94°C||94°C||55°C||68°C||--
|-
|-
-
|5min||30s||30s||30min||3cycles
+
|5min||30s||30s||1min||30cycles
|}
|}
-
[[File:igku_Aug19electrophoresis1.png]]
+
[[File:igku_Sep2electrophoresis2.png]]
</div>
</div>

Revision as of 06:47, 23 September 2013

Contents

Sep 2

Liquid Culture

Hirano

Samplemedium
9/1 entA-(Master Plate)-14mL SOB(+Km)

37°C

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

File:Igku sep2electrophoresis1.png

Restriction Enzyme Digestion

Nakamoto

pSB1C3-(1)EcoRISpeIXbaIPstIBufferBBufferDBSAMilliQtotal
2 cuts(E+S)7µL1µL1µL0µL0µL3µL0µL0.3µL17.7µL30µL
NC(E+S)0.3µL0µL0µL0µL0µL1µL0µL0.1µL8.6µL10µL
2 cut(X+P)s7µL0µL0µL1µL1µL0µL3µL0.3µL17.7µL30µL
NC(X+P)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(E+S)8µL1µL1µL0µL0µL3µL0µL0.3µL16.3µL30µL
NC(E+S)0.5µL0µL0µL0µL0µL1µL0µL0.1µL8.4µL10µL
2 cut(X+P)s8µL0µL0µL1µL1µL0µL3µL0.3µL16.3µL30µL
NC(X+P)0.5µL0µL0µL0µL0µL0µL1µL0.1µL8.4µL10µL

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

Gel Extraction

No name

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

File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
No name

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

File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx

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

File:Igku sep2electrophoresis1.png

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

File:Igku Sep2electrophoresis2.png

Master Plate

Hirano

Liquid Culture

Hirano

Colony PCR

Hirano

Transformation

Hirano
Retrieved from "http://2013.igem.org/Template:Kyoto/Notebook/Sep_2"