Template:Kyoto/Notebook/Aug 20

From 2013.igem.org

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<span class="author">Kojima and Ashida</span>
<span class="author">Kojima and Ashida</span>
{|class="wikitable"  
{|class="wikitable"  
-
!state||8/20 Ptet -1(220&micro;g/mL)!!EcoRI!!XbaI!!10x BSA!!10x buffer M!!MilliQ!!total
+
! ||8/20 Ptet -1(220&micro;g/mL)!!EcoRI!!XbaI!!10x BSA!!10x buffer M!!MilliQ!!total
|-
|-
|2 cuts||4.5||1||1||3||3||17.5||30
|2 cuts||4.5||1||1||3||3||17.5||30
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|}
|}
{|class="wikitable"  
{|class="wikitable"  
-
!state||8/20 RBS-tetR-DT -2(182&micro;g/mL)!!EcoRI!!SpeI!!10x buffer H!!MilliQ!!total
+
! ||8/20 RBS-tetR-DT -2(182&micro;g/mL)!!EcoRI!!SpeI!!10x buffer H!!MilliQ!!total
|-
|-
|2 cuts||5.5||1||1||3||19.5||30
|2 cuts||5.5||1||1||3||19.5||30
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|}
|}
{|class="wikitable"  
{|class="wikitable"  
-
!state||8/20 J23100-RBS-GFP-DT(334&micro;g/ml)!!EcoRI!!SpeI!!10x buffer H!!MilliQ!!total
+
! ||8/20 J23100-RBS-GFP-DT(334&micro;g/ml)!!EcoRI!!SpeI!!10x buffer H!!MilliQ!!total
|-
|-
|2 cuts||3.0||1||1||3||22||30
|2 cuts||3.0||1||1||3||22||30
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|}
|}
{|class="wikitable"  
{|class="wikitable"  
-
!state||8/20 J23100-RBS-luxR-DT -2(344&micro;g/mL)!!EcoRI!!XbaI!!BSA!!10x buffer M!!MilliQ!!total
+
! ||8/20 J23100-RBS-luxR-DT -2(344&micro;g/mL)!!EcoRI!!XbaI!!BSA!!10x buffer M!!MilliQ!!total
|-
|-
|2 cuts||2.9||1||1||3||3||19.1||30
|2 cuts||2.9||1||1||3||3||19.1||30
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|}
|}
{|class="wikitable"  
{|class="wikitable"  
-
!state||8/20 RBS-lysis3 -1(282&micro;g/mL)!!EcoRI!!SpeI!!10x buffer H!!MilliQ!!total
+
! ||8/20 RBS-lysis3 -1(282&micro;g/mL)!!EcoRI!!SpeI!!10x buffer H!!MilliQ!!total
|-
|-
|2 cuts||3.5||1||1||3||21.5||30
|2 cuts||3.5||1||1||3||21.5||30
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|}
|}
{|class="wikitable"  
{|class="wikitable"  
-
!state||8/17 DT -1(188&micro;g/mL)!!EcoRI!!XbaI!!10x BSA!!10x buffer M!!MilliQ!!total
+
! ||8/17 DT -1(188&micro;g/mL)!!EcoRI!!XbaI!!10x BSA!!10x buffer M!!MilliQ!!total
|-
|-
|2 cuts||5.3||1||1||3||3||16.7||30
|2 cuts||5.3||1||1||3||3||16.7||30
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|27||8/17 DT||--||--
|27||8/17 DT||--||--
|}
|}
 +
[[File:Igku Aug20Electrophoresis(Ptet E,X).jpg]]<br>
 +
[[File:Igku Aug20 Electrophoresis2(RBS‐lysis3-1).jpg]]<br>
 +
[[File:Igku Aug20Electrophoresis3(DT・E＋X).jpg]]<br>
</div>
</div>

Latest revision as of 08:42, 24 September 2013

Contents

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 Medium Plate

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.

Ristriction Enzyme Digestion

Kojima and Ashida

8/20 Ptet -1(220µg/mL)EcoRIXbaI10x BSA10x buffer MMilliQtotal
2 cuts4.5113317.530
1 cut0.50.20117.310
1 cut0.500.2117.310
NC0.500117.510
8/20 RBS-tetR-DT -2(182µg/mL)EcoRISpeI10x buffer HMilliQtotal
2 cuts5.511319.530
1 cut0.60.2018.210
1 cut0.600.218.210
NC0.60018.410
8/20 J23100-RBS-GFP-DT(334µg/ml)EcoRISpeI10x buffer HMilliQtotal
2 cuts3.01132230
1 cut0.30.2018.510
1 cut0.300.218.510
NC0.30018.710
8/20 J23100-RBS-luxR-DT -2(344µg/mL)EcoRIXbaIBSA10x buffer MMilliQtotal
2 cuts2.9113319.130
1 cut0.30.20117.510
1 cut0.300.2117.510
NC0.300117.710
8/20 RBS-lysis3 -1(282µg/mL)EcoRISpeI10x buffer HMilliQtotal
2 cuts3.511321.530
1 cut0.40.2018.410
1 cut0.400.218.410
NC0.40018.610
8/17 DT -1(188µg/mL)EcoRIXbaI10x BSA10x buffer MMilliQtotal
2 cuts5.3113316.730
1 cut0.30.20117.510
1 cut0.300.2117.510
NC0.300117.710

Transformation

Nakamoto

NameSampleCompetent Cells(XL10-gold)TotalPlate
tRNA-Spinach-tRNA1µL10µL11µLAmp
tetR-aptamer 12_PC(076)1µL10µL11µLAmp
tetR-aptamer 12_1R(113)1µL10µL11µLAmp
tetR-aptamer 12_1M(105)1µL10µL11µLAmp
pT181 attenuator1µL10µL11µLAmp
Fusin1 attenuator1µL10µL11µLAmp
Fusion3m2 attenuator1µL10µL11µLAmp
pT181 antisense1µL10µL11µLAmp
Fusion1 antisense1µL10µL11µLAmp
Fusion6 antisense1µL10µL11µLAmp

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----

Igku Aug20Electrophoresis(Ptet E,X).jpg
Igku Aug20 Electrophoresis2(RBS‐lysis3-1).jpg
Igku Aug20Electrophoresis3(DT・E＋X).jpg

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