Template:Kyoto/Notebook/Aug 22

From 2013.igem.org

(Difference between revisions)
(→Restriction Enzyme Digestion)
(→Electrophoresis)
 
(7 intermediate revisions not shown)
Line 110: Line 110:
|5min||30s||30s||1min||30cycles
|5min||30s||30s||1min||30cycles
|}
|}
 +
</div>
 +
 +
===Restriction Enzyme Digestion===
 +
<div class="experiment">
 +
<span class="author">Tatsui, Kojima, Honda and Ashida</span>
 +
{| class="wikitable"
 +
! ||Fusion1 antisense (1) (300 &micro;g/mL)||EcoRI||SpeI||buffer H||MilliQ||total
 +
|-
 +
|2 cuts||3.3||1||1||3||21.7||30
 +
|-
 +
|1 cut||0.7||0.2||0||1||8.1||10
 +
|-
 +
|1 cut||0.7||0||0.2||1||8.1||10
 +
|-
 +
|NC||0.7||0||0||1||8.3||10
 +
|}
 +
{| class="wikitable"
 +
! ||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
 +
|-
 +
|1 cut||0.7||0.2||0||1||1||7.1||10
 +
|-
 +
|1 cut||0.7||0||0.2||1||1||7.1||10
 +
|-
 +
|NC||0.7||0||0||1||1||7.3||10
 +
|}
 +
{| class="wikitable"
 +
! ||Fusion6 antisense (1) (336 &micro;g/mL)||EcoRI||SpeI||buffer H||MilliQ||total
 +
|-
 +
|2 cuts||3||1||1||3||22||30
 +
|-
 +
|1 cut||0.6||0.2||0||1||8.2||10
 +
|-
 +
|1 cut||0.6||0||0.2||1||8.2||10
 +
|-
 +
|NC||0.6||0||0||1||8.4||10
 +
|}
 +
{| class="wikitable"
 +
! ||Fusion6 antisense (1) (336 &micro;g/mL)||XbaI||PstI||buffer M||BSA||MilliQ||total
 +
|-
 +
|2 cuts||3||1||1||3||3||19||30
 +
|-
 +
|1 cut||0.6||0.2||0||1||1||7.2||10
 +
|-
 +
|1 cut||0.6||0||0.2||1||1||7.2||10
 +
|-
 +
|NC||0.6||0||0||1||1||7.4||10
 +
|}
 +
{| class="wikitable"
 +
! ||pT181 antisense (1) (178 &micro;g/mL)||EcoRI||SpeI||buffer H||MilliQ||total
 +
|-
 +
|2 cuts||5.6||1||1||3||19.4||30
 +
|-
 +
|1 cut||1.1||0.2||0||1||7.7||10
 +
|-
 +
|1 cut||1.1||0||0.2||1||7.7||10
 +
|-
 +
|NC||1.1||0||0||1||7.9||10
 +
|}
 +
{| class="wikitable"
 +
! ||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
 +
|-
 +
|1 cut||1.1||0.2||0||1||1||6.7||10
 +
|-
 +
|1 cut||1.1||0||0.2||1||1||6.7||10
 +
|-
 +
|NC||1.1||0||0||1||1||6.9||10
 +
|}
 +
{| class="wikitable"
 +
! ||pT181 attenuator (1)||EcoRI||SpeI||buffer H||MilliQ||total
 +
|-
 +
|2 cuts||3||1||1||3||22||30
 +
|-
 +
|1 cut||0.6||0.2||0||1||8.2||10
 +
|-
 +
|1 cut||0.6||0||0.2||1||8.2||10
 +
|-
 +
|NC||0.6||0||0||1||8.4||10
 +
|}
 +
{| class="wikitable"
 +
! ||pT181 attenuator (1)||XbaI||PstI||buffer M||BSA||MilliQ||total
 +
|-
 +
|2 cuts||3||1||1||3||3||19||30
 +
|-
 +
|1 cut||0.6||0.2||0||1||1||7.2||10
 +
|-
 +
|1 cut||0.6||0||0.2||1||1||7.2||10
 +
|-
 +
|NC||0.6||0||0||1||1||7.4||10
 +
|}
 +
{| class="wikitable"
 +
! ||Fusion1 attenuator (1)||EcoRI||SpeI||buffer H||MilliQ||total
 +
|-
 +
|2 cuts||3.3||1||1||3||21.7||30
 +
|-
 +
|1 cut||0.7||0.2||0||1||8.1||10
 +
|-
 +
|1 cut||0.7||0||0.2||1||8.1||10
 +
|-
 +
|NC||0.7||0||0||1||8.3||10
 +
|}
 +
{| class="wikitable"
 +
! ||Fusion1 attenuator (1)||XbaI||PstI||buffer M||BSA||MilliQ||total
 +
|-
 +
|2 cuts||3.3||1||1||3||3||18.7||30
 +
|-
 +
|1 cut||0.7||0.2||0||1||1||7.1||10
 +
|-
 +
|1 cut||0.7||0||0.2||1||1||7.1||10
 +
|-
 +
|NC||0.7||0||0||1||1||7.3||10
 +
|}
 +
{| class="wikitable"
 +
! ||Fusion3m2 attenuator (1)||EcoRI||SpeI||buffer H||MilliQ||total
 +
|-
 +
|2 cuts||3||1||1||3||22||30
 +
|-
 +
|1 cut||0.6||0.2||0||1||8.2||10
 +
|-
 +
|1 cut||0.6||0||0.2||1||8.2||10
 +
|-
 +
|NC||0.6||0||0||1||8.4||10
 +
|}
 +
{| class="wikitable"
 +
! ||Fusion3m2 attenuator (1)||XbaI||PstI||buffer M||BSA||MilliQ||total
 +
|-
 +
|2 cuts||3||1||1||3||3||19||30
 +
|-
 +
|1 cut||0.6||0.2||0||1||1||7.2||10
 +
|-
 +
|1 cut||0.6||0||0.2||1||1||7.2||10
 +
|-
 +
|NC||0.6||0||0||1||1||7.4||10
 +
|}
 +
{| class="wikitable"
 +
! ||Spinach (2)||EcoRI||SpeI||buffer B||BSA||MilliQ||total
 +
|-
 +
|2 cuts||2.3||0.5||0.5||3||0.3||23.4||30
 +
|-
 +
|1 cut||0.5||0.1||0||1||0.1||8.3||10
 +
|-
 +
|1 cut||0.5||0||0.1||1||0.1||8.3||10
 +
|-
 +
|NC||0.5||0||0||1||0.1||8.4||10
 +
|}
 +
{| class="wikitable"
 +
! ||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
 +
|-
 +
|1 cut||0.5||0.1||0||1||0.1||8.3||10
 +
|-
 +
|1 cut||0.5||0||0.1||1||0.1||8.3||10
 +
|-
 +
|NC||0.5||0||0||1||0.1||8.4||10
 +
|}
 +
{| class="wikitable"
 +
! ||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
 +
|-
 +
|1 cut||0.5||0.1||0||1||0.1||8.3||10
 +
|-
 +
|1 cut||0.5||0||0.1||1||0.1||8.3||10
 +
|-
 +
|NC||0.5||0||0||1||0.1||8.4||10
 +
|}
 +
{| class="wikitable"
 +
! ||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
 +
|-
 +
|1 cut||0.5||0.1||0||1||0.1||8.3||10
 +
|-
 +
|1 cut||0.5||0||0.1||1||0.1||8.3||10
 +
|-
 +
|NC||0.5||0||0||1||0.1||8.4||10
 +
|}
 +
{| class="wikitable"
 +
! ||J23100||SpeI||PstI||buffer B||BSA||MilliQ||total
 +
|-
 +
|2 cuts||7||0.5||0.5||3||0.3||18.7||30
 +
|-
 +
|1 cut||1.4||0.1||0||1||0.1||7.4||10
 +
|-
 +
|1 cut||1.4||0||0.1||1||0.1||7.4||10
 +
|-
 +
|NC||1.4||0||0||1||0.1||7.5||10
 +
|}
 +
{| class="wikitable"
 +
! ||Plac (2)||SpeI||PstI||buffer B||BSA||MilliQ||total
 +
|-
 +
|2 cuts||11.2||0.5||0.5||3||0.3||14.5||30
 +
|-
 +
|1 cut||2.2||0.1||0||1||0.1||6.6||10
 +
|-
 +
|1 cut||2.2||0||0.1||1||0.1||6.6||10
 +
|-
 +
|NC||2.2||0||0||1||0.1||6.7||10
 +
|}
 +
</div>
 +
===Electrophoresis===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Lane||Sample
 +
|-
 +
|1||100bp ladder
 +
|-
 +
|2||RBS-lysis1 -(1)
 +
|-
 +
|3||RBS-lysis1 -(2)
 +
|-
 +
|4||RBS-lysis2 -(1)
 +
|-
 +
|5||RBS-lysis2 -(2)
 +
|-
 +
|6||RBS(NC) -(1)
 +
|-
 +
|7||RBS(NC) -(2)
 +
|-
 +
|8||NC
 +
|}
 +
[[File:Igku Aug22 Electrophoresis(ColoP)-1.jpg]]<br>
 +
{| class="wikitable"
 +
!Lane||Sample
 +
|-
 +
|1||1kb ladder
 +
|-
 +
|2||Ptet-RBS-tetR-DT -(1)
 +
|-
 +
|3||Ptet-RBS-tetR-DT -(2)
 +
|-
 +
|4||Ptet(NC) -(1)
 +
|-
 +
|5||Ptet(NC) -(2)
 +
|-
 +
|6||Plux-RBS-GFP-DT -(1)
 +
|-
 +
|7||pSB1C3 -(1)
 +
|-
 +
|8||pSB1C3 -(2)
 +
|-
 +
|9||NC
 +
|}
 +
[[File:Igku Aug22 Electrophoresis(ColoP)-2.jpg]]<br>
 +
{| class="wikitable"
 +
!Lane||Sample
 +
|-
 +
|1||1kb ladder
 +
|-
 +
|2||Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT -(1)
 +
|-
 +
|3||Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT -(2)
 +
|-
 +
|4||Pcon-RBS-luxR -(1)
 +
|-
 +
|5||Pcon-RBS-luxR -(2)
 +
|-
 +
|6||NC
 +
|-
 +
|7||1kb ladder
 +
|}
 +
[[File:Igku Aug22 Electrophoresis(ColoP)-3.jpg]]<br>
</div>
</div>
Line 115: Line 382:
<div class="experiment">
<div class="experiment">
<span class="author">Ashida</span>
<span class="author">Ashida</span>
-
{|class="wikitable"  
+
{|class="wikitable"
-
!DNA||concentration[&micro;g/mL]||260/280||260/230  
+
!DNA||concentration[&micro;g/mL]||260/280||260/230
|-
|-
|8/22 RBS-lysis (1)||128||1.77||2.29
|8/22 RBS-lysis (1)||128||1.77||2.29

Latest revision as of 14:49, 25 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

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

Electrophoresis

No name

LaneSample
1100bp ladder
2RBS-lysis1 -(1)
3RBS-lysis1 -(2)
4RBS-lysis2 -(1)
5RBS-lysis2 -(2)
6RBS(NC) -(1)
7RBS(NC) -(2)
8NC

Igku Aug22 Electrophoresis(ColoP)-1.jpg

LaneSample
11kb ladder
2Ptet-RBS-tetR-DT -(1)
3Ptet-RBS-tetR-DT -(2)
4Ptet(NC) -(1)
5Ptet(NC) -(2)
6Plux-RBS-GFP-DT -(1)
7pSB1C3 -(1)
8pSB1C3 -(2)
9NC

Igku Aug22 Electrophoresis(ColoP)-2.jpg

LaneSample
11kb ladder
2Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT -(1)
3Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT -(2)
4Pcon-RBS-luxR -(1)
5Pcon-RBS-luxR -(2)
6NC
71kb ladder

Igku Aug22 Electrophoresis(ColoP)-3.jpg

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----

Igku Aug22electrophoresis.jpg

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----

Igku Aug22electrophoresis2.jpg

Retrieved from "http://2013.igem.org/Template:Kyoto/Notebook/Aug_22"