Template:Kyoto/Notebook/Sep 3

From 2013.igem.org

(Difference between revisions)
(→Restriction Enzyme Digestion)
(→PCR)
 
(43 intermediate revisions not shown)
Line 1: Line 1:
==Sep 3==
==Sep 3==
 +
===Electrophoresis===
===Electrophoresis===
   
   
-
<!-- こっから -->
 
<div class="experiment">
<div class="experiment">
-
<span class="author">Kozima</span>
+
<span class="author">Kojima</span>
{| class="wikitable"
{| class="wikitable"
!Lane||Sample
!Lane||Sample
Line 10: Line 10:
|1||100bp ladder
|1||100bp ladder
|-
|-
-
|2||pT181 attenuater(2)-DT2(738)
+
|2||pT181 attenuator(2)-DT2(738)
|-
|-
|3||Ptet-RBS-lacZ&alpha;-DT-2(756)
|3||Ptet-RBS-lacZ&alpha;-DT-2(756)
Line 29: Line 29:
|}
|}
-
[[File:igku_xxxxxx.xxx]]<br>
+
[[File:Igku Sep3 Electrophoresis(N1).jpg]]<br>
</div>
</div>
-
<!-- ここまでをコピーしてね -->
 
===Miniprep===
===Miniprep===
Line 47: Line 46:
|9/2 14:00 tetR-apt 12_1R||567.5||1.90||1.99  
|9/2 14:00 tetR-apt 12_1R||567.5||1.90||1.99  
|-
|-
-
|9/2 21:00 pT181 attenuater||320.9||1.90||2.15  
+
|9/2 21:00 pT181 attenuator||320.9||1.90||2.15  
|-
|-
|9/2 21:00 Spinach||457.0||1.88||2.24  
|9/2 21:00 Spinach||457.0||1.88||2.24  
Line 56: Line 55:
<!-- ここから -->
<!-- ここから -->
<div class="experiment">
<div class="experiment">
-
<span class="author">Kozima</span>
+
<span class="author">Kojima</span>
{| class="wikitable"
{| class="wikitable"
!Sample||medium
!Sample||medium
Line 67: Line 66:
===Restriction Enzyme Digestion===
===Restriction Enzyme Digestion===
<div class="experiment">
<div class="experiment">
-
<span class="author">Kozima</span>
+
<span class="author">Kojima</span>
{| class="wikitable"
{| class="wikitable"
-
! ||DNA||EcoR1||Spe1||Xba1||Pst1||10xBuffer||10xBuffer||100xBSA||MilliQ||total
+
! ||DNA||EcoRI||SpeI||XbaI||PstI||10xBuffer B(EcoRI&SpeI)||10xBuffer D(XbaI&PstI)||100xBSA||MilliQ||total
|-
|-
-
|pT181attenuater 14:00 418.4ng/&micro;L|| 4.8&micro;L|| 1.0&micro;L|| 1.0&micro;L|| 0&micro;L|| 0&micro;L|| 3&micro;L|| 0&micro;L|| 0.3&micro;L|| 19.9&micro;L|| 30&micro;L
+
|9/3 pT181attenuator 14:00 418.4ng/&micro;L|| 4.8&micro;L|| 1.0&micro;L|| 1.0&micro;L|| 0&micro;L|| 0&micro;L|| 3&micro;L|| 0&micro;L|| 0.3&micro;L|| 19.9&micro;L|| 30&micro;L
|-
|-
-
|pT181attenuater 14:00 418.4ng/&micro;L|| 0.2&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 0&micro;L|| 0.1&micro;L|| 8.7&micro;L|| 10&micro;L
+
|9/3 pT181attenuator 14:00 418.4ng/&micro;L|| 0.2&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 0&micro;L|| 0.1&micro;L|| 8.7&micro;L|| 10&micro;L
|-
|-
-
|pT181attenuater 14:00 418.4ng/&micro;L|| 4.8&micro;L|| 0&micro;L|| 0&micro;L|| 1.0&micro;L|| 1.0&micro;L|| 0&micro;L|| 3&micro;L|| 0.3&micro;L|| 19.9&micro;L|| 30&micro;L
+
|9/3 pT181attenuator 14:00 418.4ng/&micro;L|| 4.8&micro;L|| 0&micro;L|| 0&micro;L|| 1.0&micro;L|| 1.0&micro;L|| 0&micro;L|| 3&micro;L|| 0.3&micro;L|| 19.9&micro;L|| 30&micro;L
|-
|-
-
|pT181attenuater 14:00 418.4ng/&micro;L|| 0.2&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 0.1&micro;L|| 8.7&micro;L|| 10&micro;L
+
|9/3 pT181attenuator 14:00 418.4ng/&micro;L|| 0.2&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 0.1&micro;L|| 8.7&micro;L|| 10&micro;L
 +
|}
 +
</div>
 +
<div class="experiment">
 +
{| class="wikitable"
 +
! ||DNA||EcoRI||SpeI||XbaI||PstI||10xBuffer B(EcoRI&SpeI)||10xBuffer D(XbaI&PstI)||100xBSA||MilliQ||total
 +
|-
 +
|9/3 pT181antisense 14:00 362.2ng/&micro;L|| 5.5&micro;L|| 1.0&micro;L|| 1.0&micro;L|| 0&micro;L|| 0&micro;L|| 3&micro;L|| 0&micro;L|| 0.3&micro;L|| 19.2&micro;L|| 30&micro;L
 +
|-
 +
|9/3 pT181attenuator 14:00 418.4ng/&micro;L|| 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
 +
|-
 +
|9/3 pT181attenuator 14:00 418.4ng/&micro;L|| 5.5&micro;L|| 0&micro;L|| 0&micro;L|| 1.0&micro;L|| 1.0&micro;L|| 0&micro;L|| 3&micro;L|| 0.3&micro;L|| 19.2&micro;L|| 30&micro;L
 +
|-
 +
|9/3 pT181attenuator 14:00 418.4ng/&micro;L|| 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
 +
|}
 +
</div>
 +
 
 +
<div class="experiment">
 +
{| class="wikitable"
 +
! ||DNA||EcoRI||SpeI||10xBuffer B||100xBSA||MilliQ||total
 +
|-
 +
|9/3 apt 12_1R 14:00 507.5ng/&micro;L|| 3.9&micro;L|| 1.0&micro;L|| 1.0&micro;L|| 3&micro;L|| 0.3&micro;L|| 20.8&micro;L|| 30&micro;L
 +
|-
 +
|9/3 apt 12_1R 14:00 507.5ng/&micro;L|| 0.2&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 0.1&micro;L|| 8.7&micro;L|| 10&micro;L
 +
|}
 +
</div>
 +
<div class="experiment">
 +
{| class="wikitable"
 +
! ||DNA||EcoRI||XbaI||10xBuffer D||100xBSA||MilliQ||total
 +
|-
 +
|8/29 DT 166.2ng/&micro;L|| 12&micro;L|| 1.0&micro;L|| 1.0&micro;L|| 3&micro;L|| 0.3&micro;L|| 12.7&micro;L|| 30&micro;L
 +
|-
 +
|8/29 DT 166.2ng/&micro;L|| 0.6&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 0.1&micro;L|| 8.3&micro;L|| 10&micro;L
 +
|}
 +
</div>
 +
 
 +
<div class="experiment">
 +
{| class="wikitable"
 +
! ||DNA||EcoRI||SpeI||10xBuffer B||100xBSA||MilliQ||total
 +
|-
 +
|8/22 RBS-lysis1(2) 96ng/&micro;L|| 21&micro;L|| 1.0&micro;L|| 1.0&micro;L|| 3&micro;L|| 0.3&micro;L|| 3.7&micro;L|| 30&micro;L
 +
|-
 +
|8/22 RBS-lysis1(2) 96ng/&micro;L|| 1.0&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 0.1&micro;L|| 7.9&micro;L|| 10&micro;L
 +
|}
 +
</div>
 +
<div class="experiment">
 +
{| class="wikitable"
 +
! ||DNA||EcoRI||SpeI||10xBuffer B||100xBSA||MilliQ||total
 +
|-
 +
|8/30 RBS-lysis2 220ng/&micro;L|| 9.1&micro;L|| 1.0&micro;L|| 1.0&micro;L|| 3&micro;L|| 0.3&micro;L|| 15.6&micro;L|| 30&micro;L
 +
|-
 +
|8/30 RBS-lysis2 220ng/&micro;L|| 0.5&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 0.1&micro;L|| 8.4&micro;L|| 10&micro;L
 +
|}
 +
</div>
 +
 
 +
<div class="experiment">
 +
{| class="wikitable"
 +
! ||DNA||EcoRI||SpeI||10xBuffer B||100xBSA||MilliQ||total
 +
|-
 +
|8/20 RBS-lysis3(1) 282ng/&micro;L|| 7.1&micro;L|| 1.0&micro;L|| 1.0&micro;L|| 3&micro;L|| 0.3&micro;L|| 17.6&micro;L|| 30&micro;L
 +
|-
 +
|8/20 RBS-lysis3(1) 282ng/&micro;L|| 0.4&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 0.1&micro;L|| 8.5&micro;L|| 10&micro;L
|}
|}
</div>
</div>
===PCR===
===PCR===
 +
<!-- ここから -->
 +
<div class="experiment">
 +
<span class="author">Hirano</span>
 +
{| class="wikitable"
 +
!Plac(1) plasmid 153.7ng/&micro;L||10x buffer KOD plus-ver.2|||2mM dNTPs||25mM MgSO4||primer (KaiA_BsaI_Mut_fwd)||primer (KaiA_BsaI_Mut_rer)||KOD plus-ver.2||MilliQ||total
 +
|-
 +
|0.13 ||2.5||2.5||1.5||0.75||0.75||0.5||16.37||25
 +
|}
 +
</div>
 +
<div class="experiment">
 +
{| class="wikitable"
 +
!KaiB (plasmid) 20.2ng/&micro;L||10x buffer KOD plus-ver.2|||2mM dNTPs||25mM MgSO4||primer (GG2_RBS_KaiB_fwd)||primer (KaiB_GG3_rer)||KOD plus-ver.2||MilliQ||total
 +
|-
 +
|1||2.5||2.5||1.5||0.75||0.75||0.5||15.5||25
 +
|}
 +
</div>
 +
<div class="experiment">
 +
{| class="wikitable"
 +
!KaiC (plasmid) 4.4ng/&micro;L||10x buffer KOD plus-ver.2|||2mM dNTPs||25mM MgSO4||primer ||primer ||KOD plus-ver.2||MilliQ||total
 +
|-
 +
|1||2.5||2.5||1.5||0.75||0.75||0.5||15.5||25
 +
|}
 +
</div>
 +
<div class="experiment">
 +
{| class="wikitable"
 +
!DT-1 (plasmid) 153.7ng/&micro;L||10x buffer KOD plus-ver.2|||2mM dNTPs||25mM MgSO4||primer ||primer ||KOD plus-ver.2||MilliQ||total
 +
|-
 +
|0.1||2.5||2.5||1.5||0.75||0.75||0.5||16.4||25
 +
|}
 +
</div>
 +
<div class="experiment">
 +
{| class="wikitable"
 +
!PKaiBC(PCR product) 16.3ng/&micro;L||10x buffer KOD plus-ver.2|||2mM dNTPs||25mM MgSO4||primer ||primer ||KOD plus-ver.2||MilliQ||total
 +
|-
 +
|0.7||2.5||2.5||1.5||0.75||0.75||0.5||15.8||25
 +
|}
 +
</div>
 +
<div class="experiment">
 +
{| class="wikitable"
 +
!SasA(PCR product) 21.6ng/&micro;L||10x buffer KOD plus-ver.2|||2mM dNTPs||25mM MgSO4||primer ||primer ||KOD plus-ver.2||MilliQ||total
 +
|-
 +
|0.5||2.5||2.5||1.5||0.75||0.75||0.5||16||25
 +
|}
 +
</div>
 +
<div class="experiment">
 +
{| class="wikitable"
 +
!RpaA(PCR product) 15.4ng/&micro;L||10x buffer KOD plus-ver.2|||2mM dNTPs||25mM MgSO4||primer ||primer ||KOD plus-ver.2||MilliQ||total
 +
|-
 +
|0.6||2.5||2.5||1.5||0.75||0.75||0.5||15.9||25
 +
|}
 +
</div>
 +
<div class="experiment">
 +
{| class="wikitable"
 +
!RpaB(PCR product) 26.7ng/&micro;L||10x buffer KOD plus-ver.2|||2mM dNTPs||25mM MgSO4||primer ||primer ||KOD plus-ver.2||MilliQ||total
 +
|-
 +
|0.4||2.5||2.5||1.5||0.75||0.75||0.5||16.1||25
 +
|}
 +
</div>
 +
<div class="experiment">
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||98&deg;C||||68&deg;C||--
 +
|-
 +
|5min||10sec||30sec||||30
 +
|}
 +
<div class="experiment">
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||98&deg;C||||68&deg;C||--
 +
|-
 +
|5min||10sec||30sec||||30
 +
|}
 +
 +
 +
<!-- ここまでをコピーしてね -->
 +
===Mutatation PCR===
===Mutatation PCR===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!KaiABC(12.6pg/&micro;L)||Primer(Kai_BsaIMut_fwd)||Primer(Kai_BsaIMut_rer)||Prime STAR Max Premix||MilliQ||total
 +
|-
 +
|1 ||1||1||25||22||50
 +
|}
 +
{| class="wikitable"
 +
!||Primer(Kai_BsaIMut_fwd)||Primer(Kai_BsaIMut_rer)||Prime STAR Max Premix||MilliQ||total
 +
|-
 +
| ||1||1||25||23||50
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|　94&deg;C||　98&deg;C||　65&deg;C||　72&deg;C||--
 +
|-
 +
|5min||10sec　||15sec　||40sec　||30
 +
|}
 +
</div>
 +
===Colony PCR===
===Colony PCR===
-
===Restriction Enzyme Digestion===
+
<!-- ここから -->
 +
<div class="experiment">
 +
<span class="author"></span>
 +
{| class="wikitable"
 +
!Sample||base pair
 +
|-
 +
|9/1 pT181antisense(pSB1C3)||400bp
 +
|-
 +
|9/1 pT181antisense-DT||547bp
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|　94&deg;C||　94&deg;C||　55&deg;C||　68&deg;C||--
 +
|-
 +
|5m　||30s　||30s　||30s　||30
 +
|}
 +
</div>
 +
===Electrophoresis===
 +
 +
<div class="experiment">
 +
<span class="author">Kojima</span>
 +
{| class="wikitable"
 +
!Lane||Sample
 +
|-
 +
|1||100bp ladder
 +
|-
 +
|2||9/1 pT181antisense(pSB1C3)
 +
|-
 +
|3||9/1 pT181antisense
 +
|}
 +
[[File:Igku Sep3 Electrophoresis (N2).jpg]]<br>
 +
</div>
 +
 
 +
===Electrophoresis===
 +
 +
<!-- こっから -->
 +
<div class="experiment">
 +
<span class="author"></span>
 +
{| class="wikitable"
 +
!Lane||Sample
 +
|-
 +
|1 ||100bp ladder
 +
|-
 +
|2 ||attenuator EcoRI&SpeI
 +
|-
 +
|3 ||attenuator negative EcoRI&SpeI 
 +
|-
 +
|4 ||attenuator XbaI&PstI
 +
|-
 +
|5 ||attenuator negative XbaI&PstI
 +
|-
 +
|6 ||antisense EcoRI&SpeI
 +
|-
 +
|7 ||antisense negative EcoRI&SpeI
 +
|-
 +
|8 ||100bp ladder 
 +
|}
 +
 
 +
{| class="wikitable"
 +
!Lane||Sample
 +
|-
 +
|1 ||100bp ladder
 +
|-
 +
|2 ||antisense XbaI&PstI
 +
|-
 +
|3 ||antisense negative XbaI&PstI
 +
|-
 +
|4 ||apt EcoRI&SpeI
 +
|-
 +
|5 ||apt negative EcoRI&SpeI
 +
|-
 +
|6 ||DT EcoRI&XbaI
 +
|-
 +
|7 ||DT negative EcoRI&XbaI
 +
|-
 +
|8 ||100bp ladder 
 +
|}
 +
[[File:Igku Sep3 Electrophoresis(N3_2).jpg]]<br>
 +
{| class="wikitable"
 +
!Lane||Sample
 +
|-
 +
|1 ||100bp ladder
 +
|-
 +
|2 ||RBS-lysis1 EcoRI&SpeI 
 +
|-
 +
|3 ||RBS-lysis1 negative
 +
|-
 +
|4 ||RBS-lysis2 EcoRI&SpeI
 +
|-
 +
|5 ||RBS-lysis2 negative
 +
|-
 +
|6 ||RBS-lysis3 EcoRI&SpeI
 +
|-
 +
|7 ||RBS-lysis3 negative
 +
|-
 +
|8 ||100bp ladder 
 +
|}
 +
[[File:Igku Sep3 Electrophoresis(N3_3).jpg]]<br>
 +
</div>
 +
 
===Gel Extraction===
===Gel Extraction===
 +
<div class="experiment">
 +
<span class="author"></span>
 +
{| class="wikitable"
 +
!Lane||DNA||Enzyme
 +
|-
 +
|1 ||100bp ladder ||
 +
|-
 +
|3 ||pT181 attenuator||EcoRI&SpeI
 +
|-
 +
|4 ||pT181 attenuator||EcoRI&SpeI
 +
|-
 +
|6 ||pT181 attenuator||XbaI&PstI
 +
|-
 +
|7 ||pT181 attenuator||XbaI&PstI
 +
|-
 +
|9 ||pT181 antisense||EcoRI&SpeI
 +
|-
 +
|10 ||pT181 antisense||EcoRI&SpeI
 +
|}
 +
[[File:Igku Sep3 Electrophoresis(N4_1).jpg]]<br>
 +
{| class="wikitable"
 +
!Lane||DNA||Enzyme
 +
|-
 +
|1 ||100bp ladder ||
 +
|-
 +
|2 ||pT181 antisense||XbaI&PstI
 +
|-
 +
|3 ||pT181 antisense||XbaI&PstI
 +
|-
 +
|5 ||apt12_1R||EcoRI&SpeI
 +
|-
 +
|6 ||apt12_1R||EcoRI&SpeI
 +
|}
 +
[[File:Igku Sep3 Electrophoresis(N4_2).jpg]]<br>
 +
{| class="wikitable"
 +
!Lane||DNA||Enzyme
 +
|-
 +
|1 ||100bp ladder ||
 +
|-
 +
|2 ||DT||EcoRI&XbaI
 +
|-
 +
|3 ||DT||EcoRI&XbaI
 +
|-
 +
|5 ||RBS-lysis1||EcoRI&SpeI
 +
|-
 +
|6 ||RBS-lysis1||EcoRI&SpeI
 +
|}
 +
[[File:Igku Sep3 Electrophoresis(N5_3).jpg]]<br>
 +
{| class="wikitable"
 +
!Lane||DNA||Enzyme
 +
|-
 +
|1 ||100bp ladder ||
 +
|-
 +
|2 ||RBS-lysis2||EcoRI&SpeI
 +
|-
 +
|3 ||RBS-lysis2||EcoRI&SpeI
 +
|-
 +
|5 ||RBS-lysis3||EcoRI&SpeI
 +
|-
 +
|6 ||RBS-lysis3||EcoRI&SpeI
 +
|}
 +
[[File:Igku Sep3 Electrophoresis(N5_4).jpg]]<br>
 +
{| class="wikitable"
 +
!Name||concentration[&micro;g/mL]||260/280||260/230
 +
|-
 +
|pT181 attenuator(EcoRI&SpeI)||10.0 ||1.86 ||0.39
 +
|-
 +
|pT181 attenuator(XbaI&PstI) ||11.7 ||1.65 ||0.36
 +
|-
 +
|pT181 antisense(EcoRI&SpeI) ||4.2 ||1.95 ||0.25
 +
|-
 +
|pT181 antisense(XbaI&PstI)||6.6 ||1.78 ||0.33
 +
|-
 +
|tRNA 12_1R apt(EcoRI&SpeI)||5.7 ||1.73 ||0.29
 +
|-
 +
|DT(EcoRI&XbaI) ||16.6 ||1.99 ||0.76
 +
|-
 +
|RBS-lysis1(EcoRI&SpeI)||8.8 ||1.65 ||0.32
 +
|-
 +
|RBS-lysis2(EcoRI&SpeI)||13.4 ||1.61 ||0.48
 +
|-
 +
|RBS-lysis3(EcoRI&SpeI)||10.2 ||2.19 ||0.51
 +
|}
 +
</div>
 +
===Liquid Culture===
===Liquid Culture===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Sample||medium
 +
|-
 +
|8/16master plate7(DT) ||
 +
|-
 +
|8/21RBS-lysis1 ||　
 +
|}
 +
</div>

Latest revision as of 03:54, 28 September 2013

Contents

Sep 3

Electrophoresis

Kojima

LaneSample
1100bp ladder
2pT181 attenuator(2)-DT2(738)
3Ptet-RBS-lacZα-DT-2(756)
4Ptet-RBS-lacZα-DT-3
5Ptet-RBS-lacZα-DT-4
6Ptet-RBS-lacZα-DT-5
7Ptet-RBS-lacZα-DT-6
8RBS-lysis2-DT-2(985)
9RBS-lysis2-DT-3
10100bp ladder

Igku Sep3 Electrophoresis(N1).jpg

Miniprep

Tatsui

DNAconcentration[µg/mL]260/280260/230
9/2 14:00 pT181 attenuator418.41.821.80
9/2 14:00 pT181 antisense362.21.921.96
9/2 14:00 Spinach438.31.861.99
9/2 14:00 tetR-apt 12_1R567.51.901.99
9/2 21:00 pT181 attenuator320.91.902.15
9/2 21:00 Spinach457.01.882.24

Liquid Culture

Kojima

Samplemedium
Spinach-DT(9/2 master plate)plusgrow(CP)4ml

Restriction Enzyme Digestion

Kojima

DNAEcoRISpeIXbaIPstI10xBuffer B(EcoRI&SpeI)10xBuffer D(XbaI&PstI)100xBSAMilliQtotal
9/3 pT181attenuator 14:00 418.4ng/µL 4.8µL 1.0µL 1.0µL 0µL 0µL 3µL 0µL 0.3µL 19.9µL 30µL
9/3 pT181attenuator 14:00 418.4ng/µL 0.2µL 0µL 0µL 0µL 0µL 1µL 0µL 0.1µL 8.7µL 10µL
9/3 pT181attenuator 14:00 418.4ng/µL 4.8µL 0µL 0µL 1.0µL 1.0µL 0µL 3µL 0.3µL 19.9µL 30µL
9/3 pT181attenuator 14:00 418.4ng/µL 0.2µL 0µL 0µL 0µL 0µL 0µL 1µL 0.1µL 8.7µL 10µL
DNAEcoRISpeIXbaIPstI10xBuffer B(EcoRI&SpeI)10xBuffer D(XbaI&PstI)100xBSAMilliQtotal
9/3 pT181antisense 14:00 362.2ng/µL 5.5µL 1.0µL 1.0µL 0µL 0µL 3µL 0µL 0.3µL 19.2µL 30µL
9/3 pT181attenuator 14:00 418.4ng/µL 0.3µL 0µL 0µL 0µL 0µL 1µL 0µL 0.1µL 8.6µL 10µL
9/3 pT181attenuator 14:00 418.4ng/µL 5.5µL 0µL 0µL 1.0µL 1.0µL 0µL 3µL 0.3µL 19.2µL 30µL
9/3 pT181attenuator 14:00 418.4ng/µL 0.3µL 0µL 0µL 0µL 0µL 0µL 1µL 0.1µL 8.6µL 10µL
DNAEcoRISpeI10xBuffer B100xBSAMilliQtotal
9/3 apt 12_1R 14:00 507.5ng/µL 3.9µL 1.0µL 1.0µL 3µL 0.3µL 20.8µL 30µL
9/3 apt 12_1R 14:00 507.5ng/µL 0.2µL 0µL 0µL 1µL 0.1µL 8.7µL 10µL
DNAEcoRIXbaI10xBuffer D100xBSAMilliQtotal
8/29 DT 166.2ng/µL 12µL 1.0µL 1.0µL 3µL 0.3µL 12.7µL 30µL
8/29 DT 166.2ng/µL 0.6µL 0µL 0µL 1µL 0.1µL 8.3µL 10µL
DNAEcoRISpeI10xBuffer B100xBSAMilliQtotal
8/22 RBS-lysis1(2) 96ng/µL 21µL 1.0µL 1.0µL 3µL 0.3µL 3.7µL 30µL
8/22 RBS-lysis1(2) 96ng/µL 1.0µL 0µL 0µL 1µL 0.1µL 7.9µL 10µL
DNAEcoRISpeI10xBuffer B100xBSAMilliQtotal
8/30 RBS-lysis2 220ng/µL 9.1µL 1.0µL 1.0µL 3µL 0.3µL 15.6µL 30µL
8/30 RBS-lysis2 220ng/µL 0.5µL 0µL 0µL 1µL 0.1µL 8.4µL 10µL
DNAEcoRISpeI10xBuffer B100xBSAMilliQtotal
8/20 RBS-lysis3(1) 282ng/µL 7.1µL 1.0µL 1.0µL 3µL 0.3µL 17.6µL 30µL
8/20 RBS-lysis3(1) 282ng/µL 0.4µL 0µL 0µL 1µL 0.1µL 8.5µL 10µL

PCR

Hirano

Plac(1) plasmid 153.7ng/µL10x buffer KOD plus-ver.22mM dNTPs25mM MgSO4primer (KaiA_BsaI_Mut_fwd)primer (KaiA_BsaI_Mut_rer)KOD plus-ver.2MilliQtotal
0.13 2.52.51.50.750.750.516.3725
KaiB (plasmid) 20.2ng/µL10x buffer KOD plus-ver.22mM dNTPs25mM MgSO4primer (GG2_RBS_KaiB_fwd)primer (KaiB_GG3_rer)KOD plus-ver.2MilliQtotal
12.52.51.50.750.750.515.525
KaiC (plasmid) 4.4ng/µL10x buffer KOD plus-ver.22mM dNTPs25mM MgSO4primer primer KOD plus-ver.2MilliQtotal
12.52.51.50.750.750.515.525
DT-1 (plasmid) 153.7ng/µL10x buffer KOD plus-ver.22mM dNTPs25mM MgSO4primer primer KOD plus-ver.2MilliQtotal
0.12.52.51.50.750.750.516.425
PKaiBC(PCR product) 16.3ng/µL10x buffer KOD plus-ver.22mM dNTPs25mM MgSO4primer primer KOD plus-ver.2MilliQtotal
0.72.52.51.50.750.750.515.825
SasA(PCR product) 21.6ng/µL10x buffer KOD plus-ver.22mM dNTPs25mM MgSO4primer primer KOD plus-ver.2MilliQtotal
0.52.52.51.50.750.750.51625
RpaA(PCR product) 15.4ng/µL10x buffer KOD plus-ver.22mM dNTPs25mM MgSO4primer primer KOD plus-ver.2MilliQtotal
0.62.52.51.50.750.750.515.925
RpaB(PCR product) 26.7ng/µL10x buffer KOD plus-ver.22mM dNTPs25mM MgSO4primer primer KOD plus-ver.2MilliQtotal
0.42.52.51.50.750.750.516.125
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C68°C--
5min10sec30sec30
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C68°C--
5min10sec30sec30


Mutatation PCR

No name

KaiABC(12.6pg/µL)Primer(Kai_BsaIMut_fwd)Primer(Kai_BsaIMut_rer)Prime STAR Max PremixMilliQtotal
1 11252250
Primer(Kai_BsaIMut_fwd)Primer(Kai_BsaIMut_rer)Prime STAR Max PremixMilliQtotal
11252350
PreDenatureDenatureAnnealingExtensioncycle
　94°C　98°C　65°C　72°C--
5min10sec　15sec　40sec　30

Colony PCR

Samplebase pair
9/1 pT181antisense(pSB1C3)400bp
9/1 pT181antisense-DT547bp
PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　55°C　68°C--
5m　30s　30s　30s　30

Electrophoresis

Kojima

LaneSample
1100bp ladder
29/1 pT181antisense(pSB1C3)
39/1 pT181antisense

Igku Sep3 Electrophoresis (N2).jpg

Electrophoresis

LaneSample
1 100bp ladder
2 attenuator EcoRI&SpeI
3 attenuator negative EcoRI&SpeI
4 attenuator XbaI&PstI
5 attenuator negative XbaI&PstI
6 antisense EcoRI&SpeI
7 antisense negative EcoRI&SpeI
8 100bp ladder
LaneSample
1 100bp ladder
2 antisense XbaI&PstI
3 antisense negative XbaI&PstI
4 apt EcoRI&SpeI
5 apt negative EcoRI&SpeI
6 DT EcoRI&XbaI
7 DT negative EcoRI&XbaI
8 100bp ladder

Igku Sep3 Electrophoresis(N3 2).jpg

LaneSample
1 100bp ladder
2 RBS-lysis1 EcoRI&SpeI
3 RBS-lysis1 negative
4 RBS-lysis2 EcoRI&SpeI
5 RBS-lysis2 negative
6 RBS-lysis3 EcoRI&SpeI
7 RBS-lysis3 negative
8 100bp ladder

Igku Sep3 Electrophoresis(N3 3).jpg

Gel Extraction

LaneDNAEnzyme
1 100bp ladder
3 pT181 attenuatorEcoRI&SpeI
4 pT181 attenuatorEcoRI&SpeI
6 pT181 attenuatorXbaI&PstI
7 pT181 attenuatorXbaI&PstI
9 pT181 antisenseEcoRI&SpeI
10 pT181 antisenseEcoRI&SpeI

Igku Sep3 Electrophoresis(N4 1).jpg

LaneDNAEnzyme
1 100bp ladder
2 pT181 antisenseXbaI&PstI
3 pT181 antisenseXbaI&PstI
5 apt12_1REcoRI&SpeI
6 apt12_1REcoRI&SpeI

Igku Sep3 Electrophoresis(N4 2).jpg

LaneDNAEnzyme
1 100bp ladder
2 DTEcoRI&XbaI
3 DTEcoRI&XbaI
5 RBS-lysis1EcoRI&SpeI
6 RBS-lysis1EcoRI&SpeI

Igku Sep3 Electrophoresis(N5 3).jpg

LaneDNAEnzyme
1 100bp ladder
2 RBS-lysis2EcoRI&SpeI
3 RBS-lysis2EcoRI&SpeI
5 RBS-lysis3EcoRI&SpeI
6 RBS-lysis3EcoRI&SpeI

Igku Sep3 Electrophoresis(N5 4).jpg

Nameconcentration[µg/mL]260/280260/230
pT181 attenuator(EcoRI&SpeI)10.0 1.86 0.39
pT181 attenuator(XbaI&PstI) 11.7 1.65 0.36
pT181 antisense(EcoRI&SpeI) 4.2 1.95 0.25
pT181 antisense(XbaI&PstI)6.6 1.78 0.33
tRNA 12_1R apt(EcoRI&SpeI)5.7 1.73 0.29
DT(EcoRI&XbaI) 16.6 1.99 0.76
RBS-lysis1(EcoRI&SpeI)8.8 1.65 0.32
RBS-lysis2(EcoRI&SpeI)13.4 1.61 0.48
RBS-lysis3(EcoRI&SpeI)10.2 2.19 0.51

Liquid Culture

No name

Samplemedium
8/16master plate7(DT)
8/21RBS-lysis1 　
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