Team:Kyoto/Notebook

From 2013.igem.org

(Difference between revisions)
(→Aug 20)
(→Aug 19)
Line 346: Line 346:
===Miniprep===
===Miniprep===
-
==Aug 19==
+
==Aug 20==
-
===Colony PCR===
+
===Miniprep===
<div class="experiment">
<div class="experiment">
-
<span class="author">Kojima</span>
+
<span class="author">Kojima </span>
-
{| class="wikitable"
+
{|class="wikitable"  
-
!numbar||Sample||base pair
+
!DNA!!concentration[&micro;g/&micro;L]!!260/280!!260/230
|-
|-
-
|1||8/18 J23100 control -(1)||1142
+
|8/18 Ptet -1||220||1.69||1.87
|-
|-
-
|2||8/18 J23100-RBS-GFP-DT -(1)||1156
+
|8/18 Ptet -2||210||1.69||1.71
|-
|-
-
|3||8/18 J23100-RBS-GFP-DT -(2)||1156
+
|8/18 RBS-tetR-DT -1||236||1.63||1.69
|-
|-
-
|4||8/18 J23100-RBS-luxR-DT -(1)||1271
+
|8/18 RBS-tetR-DT -2||182||1.65||1.98
|-
|-
-
|5||8/18 J23100-RBS-luxR-DT -(2)||1271
+
|8/18 J23100-RBS-GFP-DT -1||334||1.71||2.12
|-
|-
-
|6||8/18 J23100-RBS-lacZ&alpha;-DT -(1)||670
+
|8/18 J23100-RBS-luxR-DT -1||440||1.67||1.60
|-
|-
-
|7||8/18 J23100-RBS-lacZ&alpha;-DT -(2)||670
+
|8/18 J23100-RBS-luxR-DT -2||344||1.68||1.83
|-
|-
-
|8||8/18 RBS-lysis3 -(1)||997
+
|8/18 J23100-RBS-lacZα-DT -1||280||1.70||1.81
|-
|-
-
|9||8/18 RBS-lysis3 -(2)||997
+
|8/18 RBS-lysis3 -1||282||1.67||1.76
|-
|-
-
|10||8/18 P0440(RBS-tetR-DT) -(1)||1154
+
|8/18 RBS-lysis3 -2||212||1.30||1.35
 +
|}
 +
</div>
 +
===LB Culture Medium===
 +
<div class="experiment">
 +
<span class="author">Okazaki</span>
 +
{|class="wikitable"
 +
!volume||200mL
|-
|-
-
|11||8/18 P0440(RBS-tetR-DT) -(2)||1154
+
|Bacto Trypton||2g
|-
|-
-
|12||negative control||-
+
|Bacto yeast extract||1g
-
|}
+
-
{| class="wikitable"
+
-
!PreDenature||Denature||Annealing||Extension||cycle
+
|-
|-
-
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||-
+
|Nacl||1g
|-
|-
-
|5min||30s||30s||1min||30cycles
+
|Agar Powder||2g
 +
|-
 +
|Amp||40&micro;l
|}
|}
-
[[File:igku_Aug19electrophoresis1.png]]
+
 
-
<br>
+
1.Measuring  reagents and put they in the Erlenmeyer flask<br>
-
{| class="wikitable"
+
2.diluting in measuring cylinder to 200ml total <br>
-
!numbar||Sample||base pair
+
3.autoclaving<br>
 +
4.adding ampicirine 40ml
 +
 
 +
 
 +
not written yet
 +
</div>
 +
===Ristriction Enzyme Digestion===
 +
<div class="experiment">
 +
<span class="author">Kojima and Ashida</span>
 +
{|class="wikitable"  
 +
!8/20 Ptet -1(220&micro;g/mL)!!EcoRI!!XbaI!!10x BSA!!10x buffer M!!MilliQ!!total
|-
|-
-
|17||8/18 RBS control -(1)||-
+
|4.5||1||1||3||3||17.5||30
|-
|-
-
|18||8/18 R0040(Ptet) -(1)||686
+
|0.5||0.2||0||1||1||7.3||10
|-
|-
-
|19||8/18 R0040(Ptet) -(2)||686
+
|0.5||0||0.2||1||1||7.3||10
|-
|-
-
|20||negative control||-
+
|0.5||0||0||1||1||7.5||10
|}
|}
-
{| class="wikitable"
+
{|class="wikitable"  
-
!PreDenature||Denature||Annealing||Extension||cycle
+
!8/20 RBS-tetR-DT -2(182&micro;g/mL)!!EcoRI!!SpeI!!10x buffer H!!MilliQ!!total
|-
|-
-
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||-
+
|5.5||1||1||3||19.5||30
|-
|-
-
|5min||30s||30s||30s||30cycles
+
|0.6||0.2||0||1||8.2||10
 +
|-
 +
|0.6||0||0.2||1||8.2||10
 +
|-
 +
|0.6||0||0||1||8.4||10
|}
|}
-
[[File:igku_Aug19electrophoresis2.png]]
+
{|class="wikitable"  
-
</div>
+
!8/20 J23100-RBS-GFP-DT(334&micro;g/ml)!!EcoRI!!SpeI!!10x buffer H!!MilliQ!!total
-
 
+
-
===Gel Extraction===
+
-
<div class="experiment">
+
-
<span class="author">Ashida</span>
+
-
{| class="wikitable"
+
-
!Lane||DNA||Enzyme
+
|-
|-
-
|1||100bp ladder||-
+
|3.0||1||1||3||22||30
|-
|-
-
|3||8/18 K117000(lysis1) -1||XbaI & PstI
+
|0.3||0.2||0||1||8.5||10
|-
|-
-
|5||8/18 K117000(lysis2) -1||XbaI & PstI
+
|0.3||0||0.2||1||8.5||10
 +
|-
 +
|0.3||0||0||1||8.7||10
|}
|}
-
[[File:igku_Aug19electrophoresis3]]<br>
+
{|class="wikitable"  
-
[[File:igku_Aug19electrophoresis4]]
+
!8/20 J23100-RBS-luxR-DT -2(344&micro;g/mL)!!EcoRI!!XbaI!!BSA!!10x buffer M!!MilliQ!!total
-
</div>
+
-
 
+
-
===LB Liquid Mideum Culture===
+
-
<!-- ここから -->
+
-
<div class="experiment">
+
-
<span class="author">Nakamoto</span>
+
-
{| class="wikitable"
+
-
!volume||200ml
+
|-
|-
-
|Bacto T2ypton || 2g
+
|2.9||1||1||3||3||19.1||30
|-
|-
-
|Bacto yeast extract ||1g
+
|0.3||0.2||0||1||1||7.5||10
|-
|-
-
|NaCl||1g
+
|0.3||0||0.2||1||1||7.5||10
|-
|-
-
|Agar Pouder||2g　
+
|0.3||0||0||1||1||7.7||10
|}
|}
-
</div>
+
{|class="wikitable"  
-
<!-- ここまでをコピーしてね -->
+
!8/20 RBS-lysis3 -1(282&micro;g/mL)!!EcoRI!!SpeI!!10x buffer H!!MilliQ!!total
-
 
+
-
===Restriction Enzyme Digestion===
+
-
<!-- こっから -->
+
-
<div class="experiment">
+
-
<span class="author">Tatsui</span>
+
-
{| class="wikitable"
+
-
!lysis2-1||XbaⅠ||PstⅠ||BSA||10xbuffer||MilliQ||total
+
|-
|-
-
|10&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||12&micro;L||30&micro;L
+
|3.5||1||1||3||21.5||30
|-
|-
-
|2&micro;L||0.2&micro;L||--||1&micro;L||1&micro;L||5.8&micro;L||10&micro;L
+
|0.4||0.2||0||1||8.4||10
|-
|-
-
|2&micro;L||--||0.2&micro;L||1&micro;L||1&micro;L||5.8&micro;L||10&micro;L
+
|0.4||0||0.2||1||8.4||10
|-
|-
-
|2&micro;LL||--||--||1&micro;L||1&micro;||6&micro;L||10&micro;L
+
|0.4||0||0||1||8.6||10
|}
|}
-
</div>
+
{|class="wikitable"  
-
*at 37&deg;C, for 1h
+
!8/17 DT -1(188&micro;g/mL)!!EcoRI!!XbaI!!10x BSA!!10x buffer M!!MilliQ!!total
-
<div class="experiment">
+
-
<span class="author">Murata and Okazaki</span>
+
-
{| class="wikitable"
+
-
!8/19 lysis1-1||10xbuffer||XbaⅠ||PstⅠ||BSA||MilliQ||total
+
|-
|-
-
|2&micro;L||2&micro;L||0.5&micro;L||0.8&micro;L||2&micro;L||13&micro;L||20&micro;L
+
|5.3||1||1||3||3||16.7||30
|-
|-
-
|0.5&micro;L||1&micro;L||0.2&micro;L||--||1&micro;L||7.3&micro;L||10&micro;L
+
|0.3||0.2||0||1||1||7.5||10
|-
|-
-
|0.5&micro;L||1&micro;L||--||0.2&micro;L||1&micro;L||7.3&micro;L||10&micro;L
+
|0.3||0||0.2||1||1||7.5||10
|-
|-
-
|0.5&micro;L||1&micro;L||--||--||1&micro;L||7.5&micro;L||10&micro;L
+
|0.3||0||0||1||1||7.7||10
|}
|}
-
*at 37℃, for 1h
+
 
 +
incubate  XX&deg;C  for Xh
</div>
</div>
-
<!-- ここまでをコピーしてね -->
 
===Transformation===
===Transformation===
-
<!-- こっから -->
 
<div class="experiment">
<div class="experiment">
-
<span class="author">Murata and Kojima</span>
+
<span class="author">Nakamoto</span>
{| class="wikitable"
{| class="wikitable"
-
!Name||Well||Sample||Competent Cells||Total||Plate
+
!Name||Sample||Competent Cells(XL10-gold)||Total||Plate
|-
|-
-
|8/18 Plux-RBS-GFP-DT||ぱーつのうぇる||2&micro;L||20&micro;L||ほにゃらら&micro;L||CP
+
|tRNA-Spinach-tRNA||1&micro;L||10&micro;L||11&micro;L||8/19 LB+Amp
|-
|-
-
|8/18 Plux(NC)||   ||2&micro;L||20&micro;L|| &micro;L ||CP
+
|tetR-aptamer 12_PC(076)||1&micro;L||10&micro;L||11&micro;L||8/19 LB+Amp
|-
|-
-
|8/19 RBS+lysis2 || || 2&micro;L||20&micro;L|| &micro;L||Amp
+
|tetR-aptamer 12_1R(113)||1&micro;L||10&micro;L||11&micro;L||8/19 LB+Amp
|-
|-
-
|8/15 Pbad-araC|| || 2&micro;L||20&micro;L|| &micro;L||Kana
+
|tetR-aptamer 12_1M(105)||1&micro;L||10&micro;L||11&micro;L||8/19 LB+Amp
 +
|-
 +
|pT181 attenuator||1&micro;L||10&micro;L||11&micro;L||8/19 LB+Amp
 +
|-
 +
|Fusin1 attenuator||1&micro;L||10&micro;L||11&micro;L||8/19 LB+Amp
 +
|-
 +
|Fusion3m2 attenuator||1&micro;L||10&micro;L||11&micro;L||8/19 LB+Amp
 +
|-
 +
|pT181 antisense||1&micro;L||10&micro;L||11&micro;L||8/19 LB+Amp
 +
|-
 +
|Fusion1 antisense||1&micro;L||10&micro;L||11&micro;L||8/19 LB+Amp
 +
|-
 +
|Fusion6 antisense||1&micro;L||10&micro;L||11&micro;L||8/19 LB+Amp
|}
|}
 +
incubate 37&deg;C overnight 20:34~
</div>
</div>
-
*Incubate at 17&deg;C
+
===Electrophoresis===
-
 
+
-
<!-- ここまでをコピーしてね -->
+
-
 
+
-
===Gel Extraction===
+
-
===Master Plate===
+
<div class="experiment">
<div class="experiment">
-
<span class="author">Ashida</span>
+
<span class="author">Kojima</span>
{| class="wikitable"
{| class="wikitable"
-
!Use LB plate(+Amp)
+
!Lane||Sample||Enzyme1||Enzyme2
|-
|-
-
|8/18 J23100-6pp(Amp)
+
|1||8/20 Ptet -1||EcoRI||XbaI
|-
|-
-
|8/18 J23100-luxR(Amp)
+
|2||8/20 Ptet -1||EcoRI||-
|-
|-
-
|8/18 J23100-luxR(Amp)
+
|3||8/20 Ptet -1||-||XbaI
|-
|-
-
|8/18 J23100+LacZα(Amp)
+
|4||8/20 Ptet -1||-||-
|-
|-
-
|8/18 RBS-lysis3(Amp)
+
|5||8/20 RBS-tetR-DT -2||EcoRI||SpeI
|-
|-
-
|8/18 RBS-lysis3(Amp)
+
|6||8/20 RBS-tetR-DT -2||EcoRI||-
-
|}
+
-
 
+
-
{| class="wikitable"
+
-
!Use LB plate(+CP)
+
|-
|-
-
|8/18 Ptet
+
|7||8/20 RBS-tetR-DT -2||-||SpeI
|-
|-
-
|8/18 RBS-tetR-DT
+
|8||8/20 RBS-tetR-DT -2||-||-
|-
|-
-
|8/18 RBS-tetR-DT
+
|9||1kbp ladder||-||-
-
|}
+
|-
-
 
+
|10||8/20 J23100-RBS-GFP-DT -1||EcoRI||SpeI
 +
|-
 +
|11||8/20 J23100-RBS-GFP-DT -1||EcoRI||-
 +
|-
 +
|12||8/20 J23100-RBS-GFP-DT -1||-||SpeI
 +
|-
 +
|13||8/20 J23100-RBS-GFP-DT -1||-||-
 +
|-
 +
|14||8/20 J23100-RBS-luxR-DT -1||EcoRI||XbaI
 +
|-
 +
|15||8/20 J23100-RBS-luxR-DT -1||EcoRI||-
 +
|-
 +
|16||8/20 J23100-RBS-luxR-DT -1||-||XbaI
 +
|-
 +
|17||8/20 J23100-RBS-luxR-DT -1||-||-
 +
|-
 +
|18||1kbp ladder||-||-
 +
|-
 +
|19||8/20 RBS-lysis3 -1||EcoRI||SpeI
 +
|-
 +
|20||8/20 RBS-lysis3 -1||EcoRI||-
 +
|-
 +
|21||8/20 RBS-lysis3 -1||-||SpeI
 +
|-
 +
|22||8/20 RBS-lysis3 -1||-||-
 +
|-
 +
|23||1kbp ladder||-||-
 +
|-
 +
|24||8/17 DT||EcoRI||XbaI
 +
|-
 +
|25||8/17 DT||EcoRI||-
 +
|-
 +
|26||8/17 DT||-||XbaI
 +
|-
 +
|27||8/17 DT||-||-
 +
|}
</div>
</div>
-
<!-- ここまでをコピーしてね -->
 
-
 
-
===Miniprep===
 
==Aug 21(changing)==
==Aug 21(changing)==

Revision as of 04:28, 4 September 2013

Home Team Official Team Profile Project Parts Submitted to the Registry Modeling Notebook Material and method Safety Attributions

Contents

template

Transformation

じっけんしゃ

NameWellSampleCompetent CellsTotalPlate
いでんしぱーつのうぇるなんちゃらµLごにょごにょµLほにゃららµLこーせーぶっしつ
いかどうよう

Restriction Enzyme Digestion

じっけんしゃ

DNABufferrestricion enzymeµLrestriction enzyme µLMilliQ µLtotal
（°°）

Electrophoresis

じっけんしゃ

DNABufferrestricion enzymeµLrestriction enzyme µLMilliQ µLtotal
（°Д°）

Ligation

じっけんしゃ

Vecter(name & concentration)quantity µL
(＞o＜)

Gel Extraction

じっけんしゃ

LaneDNAEnzyme
（＞ω・）b
Namequantityconcentration280/260260/230
（´ω｀）
（ΦωΦ^）

Colony PCR

No name

Samplebase pair
(｀・ω・´)
PreDenatureDenatureAnnealingExtensioncycle
　°C　°C　°C　°C-
（◎Д◎）　　　　

Liquid Culture

No name

Samplemedium
(・ω・)
（・Ｘ・） 　

Miniprep

DNAconcentrationµg/µL260/280260/230
(・▽・)　　　

Master Plate

No name

  • material



Aug 19

Colony PCR

Kojima

numbarSamplebase pair
18/18 J23100 control -(1)1142
28/18 J23100-RBS-GFP-DT -(1)1156
38/18 J23100-RBS-GFP-DT -(2)1156
48/18 J23100-RBS-luxR-DT -(1)1271
58/18 J23100-RBS-luxR-DT -(2)1271
68/18 J23100-RBS-lacZα-DT -(1)670
78/18 J23100-RBS-lacZα-DT -(2)670
88/18 RBS-lysis3 -(1)997
98/18 RBS-lysis3 -(2)997
108/18 P0440(RBS-tetR-DT) -(1)1154
118/18 P0440(RBS-tetR-DT) -(2)1154
12negative control-
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C-
5min30s30s1min30cycles

File:Igku Aug19electrophoresis1.png

numbarSamplebase pair
178/18 RBS control -(1)-
188/18 R0040(Ptet) -(1)686
198/18 R0040(Ptet) -(2)686
20negative control-
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C-
5min30s30s30s30cycles

File:Igku Aug19electrophoresis2.png

Gel Extraction

Ashida

LaneDNAEnzyme
1100bp ladder-
38/18 K117000(lysis1) -1XbaI & PstI
58/18 K117000(lysis2) -1XbaI & PstI

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

LB Liquid Mideum Culture

Nakamoto

volume200ml
Bacto T2ypton 2g
Bacto yeast extract 1g
NaCl1g
Agar Pouder2g　

Restriction Enzyme Digestion

Tatsui

lysis2-1XbaⅠPstⅠBSA10xbufferMilliQtotal
10µL1µL1µL3µL3µL12µL30µL
2µL0.2µL--1µL1µL5.8µL10µL
2µL--0.2µL1µL1µL5.8µL10µL
2µLL----1µL1µ6µL10µL
  • at 37°C, for 1h

Murata and Okazaki

8/19 lysis1-110xbufferXbaⅠPstⅠBSAMilliQtotal
2µL2µL0.5µL0.8µL2µL13µL20µL
0.5µL1µL0.2µL--1µL7.3µL10µL
0.5µL1µL--0.2µL1µL7.3µL10µL
0.5µL1µL----1µL7.5µL10µL
  • at 37℃, for 1h

Transformation

Murata and Kojima

NameWellSampleCompetent CellsTotalPlate
8/18 Plux-RBS-GFP-DTぱーつのうぇる2µL20µLほにゃららµLCP
8/18 Plux(NC) 2µL20µL µL CP
8/19 RBS+lysis2 2µL20µL µLAmp
8/15 Pbad-araC 2µL20µL µLKana
  • Incubate at 17°C


Gel Extraction

Master Plate

Ashida

Use LB plate(+Amp)
8/18 J23100-6pp(Amp)
8/18 J23100-luxR(Amp)
8/18 J23100-luxR(Amp)
8/18 J23100+LacZα(Amp)
8/18 RBS-lysis3(Amp)
8/18 RBS-lysis3(Amp)
Use LB plate(+CP)
8/18 Ptet
8/18 RBS-tetR-DT
8/18 RBS-tetR-DT

Miniprep

Aug 20

Miniprep

Kojima

DNAconcentration[µg/µL]260/280260/230
8/18 Ptet -12201.691.87
8/18 Ptet -22101.691.71
8/18 RBS-tetR-DT -12361.631.69
8/18 RBS-tetR-DT -21821.651.98
8/18 J23100-RBS-GFP-DT -13341.712.12
8/18 J23100-RBS-luxR-DT -14401.671.60
8/18 J23100-RBS-luxR-DT -23441.681.83
8/18 J23100-RBS-lacZα-DT -12801.701.81
8/18 RBS-lysis3 -12821.671.76
8/18 RBS-lysis3 -22121.301.35

LB Culture Medium

Okazaki

volume200mL
Bacto Trypton2g
Bacto yeast extract1g
Nacl1g
Agar Powder2g
Amp40µl

1.Measuring reagents and put they in the Erlenmeyer flask
2.diluting in measuring cylinder to 200ml total
3.autoclaving
4.adding ampicirine 40ml


not written yet

Ristriction Enzyme Digestion

Kojima and Ashida

8/20 Ptet -1(220µg/mL)EcoRIXbaI10x BSA10x buffer MMilliQtotal
4.5113317.530
0.50.20117.310
0.500.2117.310
0.500117.510
8/20 RBS-tetR-DT -2(182µg/mL)EcoRISpeI10x buffer HMilliQtotal
5.511319.530
0.60.2018.210
0.600.218.210
0.60018.410
8/20 J23100-RBS-GFP-DT(334µg/ml)EcoRISpeI10x buffer HMilliQtotal
3.01132230
0.30.2018.510
0.300.218.510
0.30018.710
8/20 J23100-RBS-luxR-DT -2(344µg/mL)EcoRIXbaIBSA10x buffer MMilliQtotal
2.9113319.130
0.30.20117.510
0.300.2117.510
0.300117.710
8/20 RBS-lysis3 -1(282µg/mL)EcoRISpeI10x buffer HMilliQtotal
3.511321.530
0.40.2018.410
0.400.218.410
0.40018.610
8/17 DT -1(188µg/mL)EcoRIXbaI10x BSA10x buffer MMilliQtotal
5.3113316.730
0.30.20117.510
0.300.2117.510
0.300117.710

incubate XX°C for Xh

Transformation

Nakamoto

NameSampleCompetent Cells(XL10-gold)TotalPlate
tRNA-Spinach-tRNA1µL10µL11µL8/19 LB+Amp
tetR-aptamer 12_PC(076)1µL10µL11µL8/19 LB+Amp
tetR-aptamer 12_1R(113)1µL10µL11µL8/19 LB+Amp
tetR-aptamer 12_1M(105)1µL10µL11µL8/19 LB+Amp
pT181 attenuator1µL10µL11µL8/19 LB+Amp
Fusin1 attenuator1µL10µL11µL8/19 LB+Amp
Fusion3m2 attenuator1µL10µL11µL8/19 LB+Amp
pT181 antisense1µL10µL11µL8/19 LB+Amp
Fusion1 antisense1µL10µL11µL8/19 LB+Amp
Fusion6 antisense1µL10µL11µL8/19 LB+Amp

incubate 37°C overnight 20:34~

Electrophoresis

Kojima

LaneSampleEnzyme1Enzyme2
18/20 Ptet -1EcoRIXbaI
28/20 Ptet -1EcoRI-
38/20 Ptet -1-XbaI
48/20 Ptet -1--
58/20 RBS-tetR-DT -2EcoRISpeI
68/20 RBS-tetR-DT -2EcoRI-
78/20 RBS-tetR-DT -2-SpeI
88/20 RBS-tetR-DT -2--
91kbp ladder--
108/20 J23100-RBS-GFP-DT -1EcoRISpeI
118/20 J23100-RBS-GFP-DT -1EcoRI-
128/20 J23100-RBS-GFP-DT -1-SpeI
138/20 J23100-RBS-GFP-DT -1--
148/20 J23100-RBS-luxR-DT -1EcoRIXbaI
158/20 J23100-RBS-luxR-DT -1EcoRI-
168/20 J23100-RBS-luxR-DT -1-XbaI
178/20 J23100-RBS-luxR-DT -1--
181kbp ladder--
198/20 RBS-lysis3 -1EcoRISpeI
208/20 RBS-lysis3 -1EcoRI-
218/20 RBS-lysis3 -1-SpeI
228/20 RBS-lysis3 -1--
231kbp ladder--
248/17 DTEcoRIXbaI
258/17 DTEcoRI-
268/17 DT-XbaI
278/17 DT--

Aug 21(changing)

Miniprep

Kojima

DNAconcentration[µg/µL]260/280260/230
8/18 Ptet -12201.691.87
8/18 Ptet -22101.691.71
8/18 RBS-tetR-DT -12361.631.69
8/18 RBS-tetR-DT -21821.651.98
8/18 J23100-RBS-GFP-DT -13341.712.12
8/18 J23100-RBS-luxR-DT -14401.671.60
8/18 J23100-RBS-luxR-DT -23441.681.83
8/18 J23100-RBS-lacZα-DT -12801.701.81
8/18 RBS-lysis3 -12821.671.76
8/18 RBS-lysis3 -22121.301.35

LB Culture Medium

Okazaki

volume200mL
Bacto Trypton2g
Bacto yeast extract1g
Nacl1g
Agar Powder2g
Amp40µl

1.Measuring reagents and put they in the Erlenmeyer flask
2.diluting in measuring cylinder to 200ml total
3.autoclaving
4.adding ampicirine 40ml


not written yet

Ristriction Enzyme Digestion

Kojima and Ashida

8/20 Ptet -1(220µg/mL)EcoRIXbaI10x BSA10x buffer MMilliQtotal
4.5113317.530
0.50.20117.310
0.500.2117.310
0.500117.510
8/20 RBS-tetR-DT -2(182µg/mL)EcoRISpeI10x buffer HMilliQtotal
5.511319.530
0.60.2018.210
0.600.218.210
0.60018.410
8/20 J23100-RBS-GFP-DT(334µg/ml)EcoRISpeI10x buffer HMilliQtotal
3.01132230
0.30.2018.510
0.300.218.510
0.30018.710
8/20 J23100-RBS-luxR-DT -2(344µg/mL)EcoRIXbaIBSA10x buffer MMilliQtotal
2.9113319.130
0.30.20117.510
0.300.2117.510
0.300117.710
8/20 RBS-lysis3 -1(282µg/mL)EcoRISpeI10x buffer HMilliQtotal
3.511321.530
0.40.2018.410
0.400.218.410
0.40018.610
8/17 DT -1(188µg/mL)EcoRIXbaI10x BSA10x buffer MMilliQtotal
5.3113316.730
0.30.20117.510
0.300.2117.510
0.300117.710

incubate XX°C for Xh

Transformation

Nakamoto

NameSampleCompetent Cells(XL10-gold)TotalPlate
tRNA-Spinach-tRNA1µL10µL11µL8/19 LB+Amp
tetR-aptamer 12_PC(076)1µL10µL11µL8/19 LB+Amp
tetR-aptamer 12_1R(113)1µL10µL11µL8/19 LB+Amp
tetR-aptamer 12_1M(105)1µL10µL11µL8/19 LB+Amp
pT181 attenuator1µL10µL11µL8/19 LB+Amp
Fusin1 attenuator1µL10µL11µL8/19 LB+Amp
Fusion3m2 attenuator1µL10µL11µL8/19 LB+Amp
pT181 antisense1µL10µL11µL8/19 LB+Amp
Fusion1 antisense1µL10µL11µL8/19 LB+Amp
Fusion6 antisense1µL10µL11µL8/19 LB+Amp

incubate 37°C overnight 20:34~

Electrophoresis

Kojima

LaneSampleEnzyme1Enzyme2
18/20 Ptet -1EcoRIXbaI
28/20 Ptet -1EcoRI-
38/20 Ptet -1-XbaI
48/20 Ptet -1--
58/20 RBS-tetR-DT -2EcoRISpeI
68/20 RBS-tetR-DT -2EcoRI-
78/20 RBS-tetR-DT -2-SpeI
88/20 RBS-tetR-DT -2--
91kbp ladder--
108/20 J23100-RBS-GFP-DT -1EcoRISpeI
118/20 J23100-RBS-GFP-DT -1EcoRI-
128/20 J23100-RBS-GFP-DT -1-SpeI
138/20 J23100-RBS-GFP-DT -1--
148/20 J23100-RBS-luxR-DT -1EcoRIXbaI
158/20 J23100-RBS-luxR-DT -1EcoRI-
168/20 J23100-RBS-luxR-DT -1-XbaI
178/20 J23100-RBS-luxR-DT -1--
181kbp ladder--
198/20 RBS-lysis3 -1EcoRISpeI
208/20 RBS-lysis3 -1EcoRI-
218/20 RBS-lysis3 -1-SpeI
228/20 RBS-lysis3 -1--
231kbp ladder--
248/17 DTEcoRIXbaI
258/17 DTEcoRI-
268/17 DT-XbaI
278/17 DT--

Aug 21

Plusgrow medium(+Amp)

Tatsui

  • material
  • 8/17 Plusgrow medium 50ml
  • 7/22 5000x　Ampicillin 10µl


  1. Measuring materials and putting them in a 50ml tube

Liquid culture

Tatsui

Samplemedium
8/20 tRNA-spinach8/21 Plusgrow medium(+Amp)
8/20 TetR-aptamer 12_P(1076)8/21 Plusgrow medium(+Amp)
8/20 TetR-aptamer 12_1R(113)8/21 Plusgrow medium(+Amp)
8/20 TetR-aptamer 12_1M(105) 8/21 Plusgrow medium(+Amp)
8/20 PT181 attenuator8/21 Plusgrow medium(+Amp)
8/20 Fusion1 attenuator 8/21 Plusgrow medium(+Amp)
8/20 Fusion3m2 attenuator 8/21 Plusgrow medium(+Amp)
8/20 PT181 antisense 8/21 Plusgrow medium(+Amp)
8/20 Fusion1 antisense 8/21 Plusgrow medium(+Amp)
8/20 Fusion6 antisense 8/21 Plusgrow medium(+Amp)

incubate 37°C 10hour

Ristriction Enzyme Digestion

Kojima and Nakamoto

8/20 Ptet-(1) EcoRIXbaI10x BSA10x buffer MMilliQtotal
4.5113317.530
0.50.20117.310
0.500.2117.310
0.500117.510
8/20 Pconst-RBS-luxR-DT-(2) EcoRIXbaI10x BSA10x buffer MMilliQtotal
2.9113319.130
0.30.20117.510
0.300.2117.510
0.300117.710
8/17 DTEcoRIXbaI10x BSA10x buffer MMilliQtotal
5.3113316.730
0.50.20117.310
0.500.2117.310
0.500117.510


incubate 37°C 1h

Electrophoresis

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-
5min30s30s30s30cycles
Samplebase pair
8/21 Ptet(pm)-RBS-tetR-DT (1)
8/21 Ptet(pm)-RBS-tetR-DT (2)
8/21 Ptet(pm) control (1)
8/21 Ptet(pm) control (2)
8/21 Plux-RBS-GFP-DT (1)
8/21 pSB1C3(BBa_J04450) (1)
8/21 pSB1C3(BBa_J04450) (2)
negative control
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C-
5min30s30s30s30cycles
==Aug 23==
Retrieved from "http://2013.igem.org/Team:Kyoto/Notebook"