Template:Kyoto/Notebook/Aug 22

From 2013.igem.org

(Difference between revisions)
(→Restriction Enzyme Digestion)
(→Restriction Enzyme Digestion)
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<span class="author">Tatsui, Kojima, Honda and Ashida</span>
<span class="author">Tatsui, Kojima, Honda and Ashida</span>
{| class="wikitable"
{| class="wikitable"
-
!state||Fusion1 antisense (1) (300 &micro;g/mL)||EcoRI||SpeI||buffer H||MilliQ||total
+
! ||Fusion1 antisense (1) (300 &micro;g/mL)||EcoRI||SpeI||buffer H||MilliQ||total
|-
|-
|2 cuts||3.3||1||1||3||21.7||30
|2 cuts||3.3||1||1||3||21.7||30
Line 212: Line 212:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
!state||Fusion1 antisense (1) (300 &micro;g/mL)||XbaI||PstI||buffer M||BSA||MilliQ||total
+
! ||Fusion1 antisense (1) (300 &micro;g/mL)||XbaI||PstI||buffer M||BSA||MilliQ||total
|-
|-
|2 cuts||3.3||1||1||3||3||18.7||30
|2 cuts||3.3||1||1||3||3||18.7||30
Line 223: Line 223:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
!state||Fusion6 antisense (1) (336 &micro;g/mL)||EcoRI||SpeI||buffer H||MilliQ||total
+
! ||Fusion6 antisense (1) (336 &micro;g/mL)||EcoRI||SpeI||buffer H||MilliQ||total
|-
|-
|2 cuts||3||1||1||3||22||30
|2 cuts||3||1||1||3||22||30
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|}
|}
{| class="wikitable"
{| class="wikitable"
-
!state||Fusion6 antisense (1) (336 &micro;g/mL)||XbaI||PstI||buffer M||BSA||MilliQ||total
+
! ||Fusion6 antisense (1) (336 &micro;g/mL)||XbaI||PstI||buffer M||BSA||MilliQ||total
|-
|-
|2 cuts||3||1||1||3||3||19||30
|2 cuts||3||1||1||3||3||19||30
Line 245: Line 245:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
!state||pT181 antisense (1) (178 &micro;g/mL)||EcoRI||SpeI||buffer H||MilliQ||total
+
! ||pT181 antisense (1) (178 &micro;g/mL)||EcoRI||SpeI||buffer H||MilliQ||total
|-
|-
|2 cuts||5.6||1||1||3||19.4||30
|2 cuts||5.6||1||1||3||19.4||30
Line 256: Line 256:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
!state||pT181 antisense (1) (178 &micro;g/mL)||XbaI||PstI||buffer M||BSA||MilliQ||total
+
! ||pT181 antisense (1) (178 &micro;g/mL)||XbaI||PstI||buffer M||BSA||MilliQ||total
|-
|-
|2 cuts||5.6||1||1||3||3||16.4||30
|2 cuts||5.6||1||1||3||3||16.4||30
Line 267: Line 267:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
!state||pT181 attenuator (1)||EcoRI||SpeI||buffer H||MilliQ||total
+
! ||pT181 attenuator (1)||EcoRI||SpeI||buffer H||MilliQ||total
|-
|-
|2 cuts||3||1||1||3||22||30
|2 cuts||3||1||1||3||22||30
Line 278: Line 278:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
!state||pT181 attenuator (1)||XbaI||PstI||buffer M||BSA||MilliQ||total
+
! ||pT181 attenuator (1)||XbaI||PstI||buffer M||BSA||MilliQ||total
|-
|-
|2 cuts||3||1||1||3||3||19||30
|2 cuts||3||1||1||3||3||19||30
Line 289: Line 289:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
!state||Fusion1 attenuator (1)||EcoRI||SpeI||buffer H||MilliQ||total
+
! ||Fusion1 attenuator (1)||EcoRI||SpeI||buffer H||MilliQ||total
|-
|-
|2 cuts||3.3||1||1||3||21.7||30
|2 cuts||3.3||1||1||3||21.7||30
Line 300: Line 300:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
!state||Fusion1 attenuator (1)||XbaI||PstI||buffer M||BSA||MilliQ||total
+
! ||Fusion1 attenuator (1)||XbaI||PstI||buffer M||BSA||MilliQ||total
|-
|-
|2 cuts||3.3||1||1||3||3||18.7||30
|2 cuts||3.3||1||1||3||3||18.7||30
Line 311: Line 311:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
!state||Fusion3m2 attenuator (1)||EcoRI||SpeI||buffer H||MilliQ||total
+
! ||Fusion3m2 attenuator (1)||EcoRI||SpeI||buffer H||MilliQ||total
|-
|-
|2 cuts||3||1||1||3||22||30
|2 cuts||3||1||1||3||22||30
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|}
|}
{| class="wikitable"
{| class="wikitable"
-
!state||Fusion3m2 attenuator (1)||XbaI||PstI||buffer M||BSA||MilliQ||total
+
! ||Fusion3m2 attenuator (1)||XbaI||PstI||buffer M||BSA||MilliQ||total
|-
|-
|2 cuts||3||1||1||3||3||19||30
|2 cuts||3||1||1||3||3||19||30
Line 333: Line 333:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
!state||Spinach (2)||EcoRI||SpeI||buffer B||BSA||MilliQ||total
+
! ||Spinach (2)||EcoRI||SpeI||buffer B||BSA||MilliQ||total
|-
|-
|2 cuts||2.3||0.5||0.5||3||0.3||23.4||30
|2 cuts||2.3||0.5||0.5||3||0.3||23.4||30
Line 344: Line 344:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
!state||tetR aptamer 12_P (2)||EcoRI||SpeI||buffer B||BSA||MilliQ||total
+
! ||tetR aptamer 12_P (2)||EcoRI||SpeI||buffer B||BSA||MilliQ||total
|-
|-
|2 cuts||2.7||0.5||0.5||3||0.3||23||30
|2 cuts||2.7||0.5||0.5||3||0.3||23||30
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|}
|}
{| class="wikitable"
{| class="wikitable"
-
!state||tetR aptamer 12_1R (2)||EcoRI||SpeI||buffer B||BSA||MilliQ||total
+
! ||tetR aptamer 12_1R (2)||EcoRI||SpeI||buffer B||BSA||MilliQ||total
|-
|-
|2 cuts||2.6||0.5||0.5||3||0.3||23.1||30
|2 cuts||2.6||0.5||0.5||3||0.3||23.1||30
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|}
|}
{| class="wikitable"
{| class="wikitable"
-
!state||tetR aptamer 12_1M (1)||EcoRI||SpeI||buffer B||BSA||MilliQ||total
+
! ||tetR aptamer 12_1M (1)||EcoRI||SpeI||buffer B||BSA||MilliQ||total
|-
|-
|2 cuts||2.7||0.5||0.5||3||0.3||23||30
|2 cuts||2.7||0.5||0.5||3||0.3||23||30
Line 377: Line 377:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
!state||J23100||SpeI||PstI||buffer B||BSA||MilliQ||total
+
! ||J23100||SpeI||PstI||buffer B||BSA||MilliQ||total
|-
|-
|2 cuts||7||0.5||0.5||3||0.3||18.7||30
|2 cuts||7||0.5||0.5||3||0.3||18.7||30
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|}
|}
{| class="wikitable"
{| class="wikitable"
-
!state||Plac (2)||SpeI||PstI||buffer B||BSA||MilliQ||total
+
! ||Plac (2)||SpeI||PstI||buffer B||BSA||MilliQ||total
|-
|-
|2 cuts||11.2||0.5||0.5||3||0.3||14.5||30
|2 cuts||11.2||0.5||0.5||3||0.3||14.5||30

Revision as of 22:47, 20 September 2013

Contents

Aug 22

Liquid culture

Nakamoto

SampleMedium
8/21 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT (1)8/21 Plusgrow medium (+Amp)
8/21 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT (2)8/21 Plusgrow medium (+Amp)
8/21 Pcon-RBS-GFP-DT control (1)8/21 Plusgrow medium (+Amp)
8/21 Pcon-RBS-GFP-DT control (2)8/21 Plusgrow medium (+Amp)
8/21 RBS-lysis1 (1)8/21 Plusgrow medium (+Amp)
8/21 RBS-lysis1 (2)8/21 Plusgrow medium (+Amp)
8/21 RBS control (1)8/21 Plusgrow medium (+Amp)
8/21 RBS control (2)8/21 Plusgrow medium (+Amp)
8/21 Ptet(pm)-RBS-tetR-DT (1)8/21 Plusgrow medium (+Amp)
8/21 Ptet(pm)-RBS-tetR-DT (2)8/21 Plusgrow medium (+Amp)
8/21 Ptet(pm) control (1)8/21 Plusgrow medium (+Amp)
8/21 Ptet(pm) control (2)8/21 Plusgrow medium (+Amp)
8/21 Plux-RBS-GFP-DT (1)8/21 Plusgrow medium (+Amp)
8/21 pSB1C3(BBa_J04450) (1)8/21 Plusgrow medium (+Amp)
8/21 pSB1C3(BBa_J04450) (2)8/21 Plusgrow medium (+Amp)

incubate 37°C 10hour

Colony PCR

Kojima

Samplebase pair
8/21 RBS-lysis1(1)400
8/21 RBS-lysis1(2)400
8/21 RBS control(1)--
8/21 RBS control(2)--
negative control--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s30s30cycles
Samplebase pair
8/21 Pcon-GFP-DT-Pcon-RBS-luxR-DT (1)2143
8/21 Pcon-GFP-DT-Pcon-RBS-luxR-DT (2)2143
8/21 Pcon-RBS-luxR control(1)--
8/21 Pcon-RBS-luxR control(2)--
negative control--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s2min30cycles
Samplebase pair
8/21 Ptet-RBS-tetR-DT (1)1216
8/21 Ptet-RBS-tetR-DT (2)1216
8/21 Ptet control (1)--
8/21 Ptet control (2)--
8/21 Plux-RBS-GFP-DT (1)1227
8/21 pSB1C3(BBa_J04450) (1)1353
8/21 pSB1C3(BBa_J04450) (2)1353
negative control--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min30cycles

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1pT181antisenseEcoRISpeI
2pT181antisenseEcoRI--
3pT181antisense--SpeI
4pT181antisense----
5pT181antisenseXbaIPstI
6pT181antisenseXbaI--
7pT181antisense--PstI
8pT181antisense----
9100bp ladder----
10Fusion1 attenuatorEcoRISpeI
11Fusion1 attenuatorEcoRI--
12Fusion1 attenuator--SpeI
13Fusion1 attenuator----
14Fusion1 attenuatorXbaIPstI
15Fusion1 attenuatorXbaI--
16fusion1 attenuator--PstI
17fusion1 attenuator----

File:Igku xxxxxx.xxx

LaneSampleEnzyme1Enzyme2
1Fusion3 3m2 attenuatorEcoRISpeI
2Fusion3 3m2 attenuatorEcoRI--
3Fusion3 3m2 attenuator--SpeI
4Fusion3 3m2 attenuator----
5Fusion3 3m2 attenuatorXbaIPstI
6Fusion3 3m2 attenuatorXbaI--
7Fusion3 3m2 attenuator--PstI
8Fusion3 3m2 attenuator----
9100bp ladder----
10aptamer 12-1MEcoRISpeI
11aptamer 12-1MEcoRI--
12aptamer 12-1M--SpeI
13aptamer 12-1M----
14aptamer 12-PEcoRISpeI
15aptamer 12-PEcoRI--
16aptamer 12-1P--SpeI
17aptamer 12-1P----

File:Igku xxxxxx.xxx

Restriction Enzyme Digestion

Tatsui, Kojima, Honda and Ashida

Fusion1 antisense (1) (300 µg/mL)EcoRISpeIbuffer HMilliQtotal
2 cuts3.311321.730
1 cut0.70.2018.110
1 cut0.700.218.110
NC0.70018.310
Fusion1 antisense (1) (300 µg/mL)XbaIPstIbuffer MBSAMilliQtotal
2 cuts3.3113318.730
1 cut0.70.20117.110
1 cut0.700.2117.110
NC0.700117.310
Fusion6 antisense (1) (336 µg/mL)EcoRISpeIbuffer HMilliQtotal
2 cuts31132230
1 cut0.60.2018.210
1 cut0.600.218.210
NC0.60018.410
Fusion6 antisense (1) (336 µg/mL)XbaIPstIbuffer MBSAMilliQtotal
2 cuts311331930
1 cut0.60.20117.210
1 cut0.600.2117.210
NC0.600117.410
pT181 antisense (1) (178 µg/mL)EcoRISpeIbuffer HMilliQtotal
2 cuts5.611319.430
1 cut1.10.2017.710
1 cut1.100.217.710
NC1.10017.910
pT181 antisense (1) (178 µg/mL)XbaIPstIbuffer MBSAMilliQtotal
2 cuts5.6113316.430
1 cut1.10.20116.710
1 cut1.100.2116.710
NC1.100116.910
pT181 attenuator (1)EcoRISpeIbuffer HMilliQtotal
2 cuts31132230
1 cut0.60.2018.210
1 cut0.600.218.210
NC0.60018.410
pT181 attenuator (1)XbaIPstIbuffer MBSAMilliQtotal
2 cuts311331930
1 cut0.60.20117.210
1 cut0.600.2117.210
NC0.600117.410
Fusion1 attenuator (1)EcoRISpeIbuffer HMilliQtotal
2 cuts3.311321.730
1 cut0.70.2018.110
1 cut0.700.218.110
NC0.70018.310
Fusion1 attenuator (1)XbaIPstIbuffer MBSAMilliQtotal
2 cuts3.3113318.730
1 cut0.70.20117.110
1 cut0.700.2117.110
NC0.700117.310
Fusion3m2 attenuator (1)EcoRISpeIbuffer HMilliQtotal
2 cuts31132230
1 cut0.60.2018.210
1 cut0.600.218.210
NC0.60018.410
Fusion3m2 attenuator (1)XbaIPstIbuffer MBSAMilliQtotal
2 cuts311331930
1 cut0.60.20117.210
1 cut0.600.2117.210
NC0.600117.410
Spinach (2)EcoRISpeIbuffer BBSAMilliQtotal
2 cuts2.30.50.530.323.430
1 cut0.50.1010.18.310
1 cut0.500.110.18.310
NC0.50010.18.410
tetR aptamer 12_P (2)EcoRISpeIbuffer BBSAMilliQtotal
2 cuts2.70.50.530.32330
1 cut0.50.1010.18.310
1 cut0.500.110.18.310
NC0.50010.18.410
tetR aptamer 12_1R (2)EcoRISpeIbuffer BBSAMilliQtotal
2 cuts2.60.50.530.323.130
1 cut0.50.1010.18.310
1 cut0.500.110.18.310
NC0.50010.18.410
tetR aptamer 12_1M (1)EcoRISpeIbuffer BBSAMilliQtotal
2 cuts2.70.50.530.32330
1 cut0.50.1010.18.310
1 cut0.500.110.18.310
NC0.50010.18.410
J23100SpeIPstIbuffer BBSAMilliQtotal
2 cuts70.50.530.318.730
1 cut1.40.1010.17.410
1 cut1.400.110.17.410
NC1.40010.17.510
Plac (2)SpeIPstIbuffer BBSAMilliQtotal
2 cuts11.20.50.530.314.530
1 cut2.20.1010.16.610
1 cut2.200.110.16.610
NC2.20010.16.710

Miniprep

Ashida

DNAconcentration[µg/mL]260/280260/230
8/22 RBS-lysis (1)1281.772.29
8/22 RBS-lysis (2)961.802.07
8/22 pSB1C3 (1)1081.551.45
8/22 pSB1C3 (2)1301.691.76

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp----
28/22 pT181 attenuator (1)EcoRISpeI
38/22 pT181 attenuator (1)EcoRI--
48/22 pT181 attenuator (1)--SpeI
58/22 pT181 attenuator (1)----
68/22 pT181 attenuator (1)XbaIPstI
78/22 pT181 attenuator (1)XbaI--
88/22 pT181 attenuator (1)--PstI
98/22 pT181 attenuator (1)----
108/22 Fusion6 antisense (1)EcoRISpeI
118/22 Fusion6 antisense (1)EcoRI--
128/22 Fusion6 antisense (1)--SpeI
138/22 Fusion6 antisense (1)----
14100bp----

File:Igku xxxxxx.xxx

LaneSampleEnzyme1Enzyme2
1100bp----
28/22 Fusion6 antisense (1)XbaIPstI
38/22 Fusion6 antisense (1)XbaI--
48/22 Fusion6 antisense (1)--PstI
58/22 Fusion6 antisense (1)----
68/22 Fusion1 antisense (1)EcoRISpeI
78/22 Fusion1 antisense (1)EcoRI--
88/22 Fusion1 antisense (1)--SpeI
98/22 Fusion1 antisense (1)----
108/22 Fusion1 antisense (1)XbaIPstI
118/22 Fusion1 antisense (1)XbaI--
128/22 Fusion1 antisense (1)--PstI
138/22 Fusion1 antisense (1)----
14100bp----

File:Igku xxxxxx.xxx

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