Template:Kyoto/Notebook/Aug 19

From 2013.igem.org

(Difference between revisions)
(→Restriction Enzyme Digestion)
(→Liquid Culture)
 
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Line 37: Line 37:
|5min||30s||30s||1min||30cycles
|5min||30s||30s||1min||30cycles
|}
|}
-
[[File:igku_Aug19electrophoresis1.png]]
+
[[File:Igku Aug19electrophoresis1(colony PCR).png]]
<br>
<br>
 +
{| class="wikitable"
 +
!Lane||DNA
 +
|-
 +
|1||100bp ladder
 +
|-
 +
|2||8/18 NC J23100(1)
 +
|-
 +
|3||8/18 J23100-RBS-GFP-DT-(1)
 +
|-
 +
|4||8/18 J23100-RBS-GFP-DT-(2)
 +
|-
 +
|5||8/18 J23100-RBS-luxR-DT-(1)
 +
|-
 +
|6||8/18 J23100-RBS-luxR-DT-(2)
 +
|-
 +
|7||8/18 J23100-RBS-lacZ&alpha;-DT-(1)
 +
|-
 +
|8||8/18 J23100-RBS-lacZ&alpha;-DT-(2)
 +
|-
 +
|9||8/18 RBS-lysis3-(1)
 +
|-
 +
|10||8/18 RBS-lysis3-(2)
 +
|-
 +
|11||8/18 R0400(RBS-tetR-DT)-(1)
 +
|-
 +
|12||8/18 R0400(RBS-tetR-DT)-(2)
 +
|-
 +
|13||NC
 +
|-
 +
|14||100bp ladder
 +
|}
 +
 +
[[File:Igku Aug19electrophoresis1(Colony PCR).png]]<br>
 +
{| class="wikitable"
 +
!Name||concentration[&micro;g/mL]||260/280||260/230
 +
|-
 +
|8/19 lysis1 &#9312; (XbaI & PstI)||87.8||1.06||0.82
 +
|}
 +
 +
{| class="wikitable"
{| class="wikitable"
!Sample||base pair
!Sample||base pair
Line 57: Line 97:
|5min||30s||30s||30s||30cycles
|5min||30s||30s||30s||30cycles
|}
|}
-
[[File:igku_Aug19electrophoresis2.png]]
 
</div>
</div>
Line 72: Line 111:
|5||8/18 K117000(lysis2) -1||XbaI & PstI
|5||8/18 K117000(lysis2) -1||XbaI & PstI
|}
|}
-
[[File:igku_Aug19electrophoresis3]]<br>
+
[[File:igku_Aug19electrophoresis3(Gel Extraction).png]]<br>
-
[[File:igku_Aug19electrophoresis4]]<br>
+
[[File:igku_Aug19electrophoresis4(Gel Extraction).png]]<br>
{| class="wikitable"
{| class="wikitable"
!Name||concentration[&micro;g/mL]||260/280||260/230
!Name||concentration[&micro;g/mL]||260/280||260/230
Line 96: Line 135:
|NaCl||1g
|NaCl||1g
|-
|-
-
|Agar Pouder||2g　
+
|Agar Pouder||2g
|}
|}
</div>
</div>
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<span class="author">Tatsui</span>
<span class="author">Tatsui</span>
{| class="wikitable"
{| class="wikitable"
-
!state||lysis2-1||XbaI||PstI||10xbuffer||BSA||MilliQ||total
+
! ||lysis2-1||XbaI||PstI||10xbuffer||BSA||MilliQ||total
|-
|-
|2 cuts||10&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||12&micro;L||30&micro;L
|2 cuts||10&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||12&micro;L||30&micro;L
|-
|-
-
|1 cuts||2&micro;L||0.2&micro;L||--||1&micro;L||1&micro;L||5.8&micro;L||10&micro;L
+
|1 cut||2&micro;L||0.2&micro;L||--||1&micro;L||1&micro;L||5.8&micro;L||10&micro;L
|-
|-
-
|1 cuts||2&micro;L||--||0.2&micro;L||1&micro;L||1&micro;L||5.8&micro;L||10&micro;L
+
|1 cut||2&micro;L||--||0.2&micro;L||1&micro;L||1&micro;L||5.8&micro;L||10&micro;L
|-
|-
-
|NC||2&micro;LL||--||--||1&micro;L||1&micro;||6&micro;L||10&micro;L
+
|NC||2&micro;L||--||--||1&micro;L||1&micro;||6&micro;L||10&micro;L
|}
|}
*at 37&deg;C, for 1h
*at 37&deg;C, for 1h
-
<span class="author">Murata and Okazaki</span>
+
<span class="author">Murakami and Okazaki</span>
{| class="wikitable"
{| class="wikitable"
-
!state||8/19 lysis1-1||XbaI||PstI||10xbuffer||BSA||MilliQ||total
+
! ||8/19 lysis1-1||XbaI||PstI||10xbuffer||BSA||MilliQ||total
|-
|-
|2 cuts||2&micro;L||0.5&micro;L||0.8&micro;L||2&micro;L||2&micro;L||13&micro;L||20&micro;L
|2 cuts||2&micro;L||0.5&micro;L||0.8&micro;L||2&micro;L||2&micro;L||13&micro;L||20&micro;L
|-
|-
-
|1 cuts||0.5&micro;L||0.2&micro;L||--||1&micro;L||1&micro;L||7.3&micro;L||10&micro;L
+
|1 cut||0.5&micro;L||0.2&micro;L||--||1&micro;L||1&micro;L||7.3&micro;L||10&micro;L
|-
|-
-
|1 cuts||0.5&micro;L||--||0.2&micro;L||1&micro;L||1&micro;L||7.3&micro;L||10&micro;L
+
|1 cut||0.5&micro;L||--||0.2&micro;L||1&micro;L||1&micro;L||7.3&micro;L||10&micro;L
|-
|-
|NC||0.5&micro;L||--||--||1&micro;L||1&micro;L||7.5&micro;L||10&micro;L
|NC||0.5&micro;L||--||--||1&micro;L||1&micro;L||7.5&micro;L||10&micro;L
|}
|}
*at 37&deg;C, for 1h
*at 37&deg;C, for 1h
 +
[[File:Igku Aug19 Restriction Enzyme Digestion(lysis2-1(1)).jpg]]
</div>
</div>
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|6||8/19 lysis1(Gel Extraction Product)||XbaI||PstI
|6||8/19 lysis1(Gel Extraction Product)||XbaI||PstI
|}
|}
 +
[[File:Igku Aug19 Electrophoresis lysis1.jpg]]<br>
 +
</div>
</div>
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|2||8/19 lysis1 &#9312;||XbaI & PstI
|2||8/19 lysis1 &#9312;||XbaI & PstI
|}
|}
-
[fig before]
+
[[File:Igku Aug19 Gel Extraction(lysis①-1)before.jpg]]<br>
-
[fig after]
+
[[File:Igku Aug19 Gel Extraction(lysis①-1)after.jpg]]<br>
{| class="wikitable"
{| class="wikitable"
!Name||concentration[&micro;g/mL]||260/280||260/230
!Name||concentration[&micro;g/mL]||260/280||260/230
|-
|-
-
|8/19 lysis1 &#9312;(XbaI & PstI)||87.8||1.06||0.82
+
|8/19 lysis1 &#9312; (XbaI & PstI)||87.8||1.06||0.82
|}
|}
</div>
</div>
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|8/18 Ptet -2||Plusgrow medium(+CP)
|8/18 Ptet -2||Plusgrow medium(+CP)
|-
|-
-
|8/18 RBS-tetR-DT -1||Plusgrow medium(+CP)　
+
|8/18 RBS-tetR-DT -1||Plusgrow medium(+CP)
|-
|-
-
|8/18 RBS-tetR-DT -2||Plusgrow medium(+CP)　
+
|8/18 RBS-tetR-DT -2||Plusgrow medium(+CP)
|-
|-
|8/18 J23100-GFP -1||Plusgrow medium(+Amp)
|8/18 J23100-GFP -1||Plusgrow medium(+Amp)

Latest revision as of 03:44, 28 September 2013

Contents

Aug 19

Colony PCR

Kojima

Samplebase pair
8/18 J23100 control -(1)1142
8/18 J23100-RBS-GFP-DT -(1)1156
8/18 J23100-RBS-GFP-DT -(2)1156
8/18 J23100-RBS-luxR-DT -(1)1271
8/18 J23100-RBS-luxR-DT -(2)1271
8/18 J23100-RBS-lacZα-DT -(1)670
8/18 J23100-RBS-lacZα-DT -(2)670
8/18 RBS-lysis3 -(1)997
8/18 RBS-lysis3 -(2)997
8/18 P0440(RBS-tetR-DT) -(1)1154
8/18 P0440(RBS-tetR-DT) -(2)1154
negative control--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min30cycles

Igku Aug19electrophoresis1(colony PCR).png

LaneDNA
1100bp ladder
28/18 NC J23100(1)
38/18 J23100-RBS-GFP-DT-(1)
48/18 J23100-RBS-GFP-DT-(2)
58/18 J23100-RBS-luxR-DT-(1)
68/18 J23100-RBS-luxR-DT-(2)
78/18 J23100-RBS-lacZα-DT-(1)
88/18 J23100-RBS-lacZα-DT-(2)
98/18 RBS-lysis3-(1)
108/18 RBS-lysis3-(2)
118/18 R0400(RBS-tetR-DT)-(1)
128/18 R0400(RBS-tetR-DT)-(2)
13NC
14100bp ladder

Igku Aug19electrophoresis1(Colony PCR).png

Nameconcentration[µg/mL]260/280260/230
8/19 lysis1 ① (XbaI & PstI)87.81.060.82


Samplebase pair
8/18 RBS control -(1)--
8/18 R0040(Ptet) -(1)686
8/18 R0040(Ptet) -(2)686
negative control--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s30s30cycles

Gel Extraction

Ashida

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

Igku Aug19electrophoresis3(Gel Extraction).png
Igku Aug19electrophoresis4(Gel Extraction).png

Nameconcentration[µg/mL]260/280260/230
8/18 lysis2-1(XbaI & PstI)3.13.640.19
8/18 lysis2-1(XbaI & PstI)3.41.890.13

LB Medium Plate

Nakamoto

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

Restriction Enzyme Digestion

Tatsui

lysis2-1XbaIPstI10xbufferBSAMilliQtotal
2 cuts10µL1µL1µL3µL3µL12µL30µL
1 cut2µL0.2µL--1µL1µL5.8µL10µL
1 cut2µL--0.2µL1µL1µL5.8µL10µL
NC2µL----1µL1µ6µL10µL
  • at 37°C, for 1h

Murakami and Okazaki

8/19 lysis1-1XbaIPstI10xbufferBSAMilliQtotal
2 cuts2µL0.5µL0.8µL2µL2µL13µL20µL
1 cut0.5µL0.2µL--1µL1µL7.3µL10µL
1 cut0.5µL--0.2µL1µL1µL7.3µL10µL
NC0.5µL----1µL1µL7.5µL10µL
  • at 37°C, for 1h

Igku Aug19 Restriction Enzyme Digestion(lysis2-1(1)).jpg

Ligation

No name

stateVectorInserterLigation High ver.2
experimentRBS0.3lysis27.44
NCRBS0.3MilliQ7.44

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp ladder----
28/19 lysis1XbaIPstI
38/19 lysis1XbaI--
48/19 lysis1--PstI
58/19 lysis1----
68/19 lysis1(Gel Extraction Product)XbaIPstI

Igku Aug19 Electrophoresis lysis1.jpg

Transformation

Murata and Kojima

NameSampleCompetent CellsPlate
8/18 Plux+RBS-GFP-DT2µL20µLCP
8/18 Plux(NC)2µL20µLCP
8/19 RBS+lysis2 2µL20µLAmp
8/15 Pbad-araC2µL20µLKan
  • Incubate at 37°C

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder--
28/19 lysis1 ①XbaI & PstI

Igku Aug19 Gel Extraction(lysis①-1)before.jpg
Igku Aug19 Gel Extraction(lysis①-1)after.jpg

Nameconcentration[µg/mL]260/280260/230
8/19 lysis1 ① (XbaI & PstI)87.81.060.82

Liquid Culture

Ashida

Samplemedium
8/18 Ptet -1Plusgrow medium(+CP)
8/18 Ptet -2Plusgrow medium(+CP)
8/18 RBS-tetR-DT -1Plusgrow medium(+CP)
8/18 RBS-tetR-DT -2Plusgrow medium(+CP)
8/18 J23100-GFP -1Plusgrow medium(+Amp)
8/18 J23100-luxR -1Plusgrow medium(+Amp)
8/18 J23100-luxR -2Plusgrow medium(+Amp)
8/18 J23100-lacZα -1Plusgrow medium(+Amp)
8/18 RBS-lysis3 -1Plusgrow medium(+Amp)
8/18 RBS-lysis3 -2Plusgrow medium(+Amp)

Master Plate

Ashida

NumberUse LB plate(+Amp)
18/18 J23100-GFP -1
28/18 J23100-luxR -1
38/18 J23100-luxR -2
48/18 J23100-lacZα -1
58/18 RBS-lysis3 -1
68/18 RBS-lysis3 -2
NumberUse LB plate(+CP)
18/18 Ptet -1
18/18 Ptet -2
38/18 RBS-tetR-DT -1
48/18 RBS-tetR-DT -2
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