Template:Kyoto/Notebook/Sep 9

From 2013.igem.org

(Difference between revisions)
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(→PCR)
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<span class="author">Tatsui and Yoshida</span>
<span class="author">Tatsui and Yoshida</span>
{| class="wikitable"
{| class="wikitable"
-
!Kai(100pg)||10x buffer KOD plus ver2||2M dNTP||MgSO4||Primer Mix(F&R)||Kod-plus ver2||MilliQ||total
+
!KaiC||10x buffer KOD plus ver2||2M dNTP||MgSO4||Primer Mix(F&R)||Kod-plus ver2||MilliQ||total
|-
|-
|0.5||2.5||2.5||1.5||1.5||0.5||16.0||25.0
|0.5||2.5||2.5||1.5||1.5||0.5||16.0||25.0
-
|}
 
-
{| class="wikitable"
 
-
!PreDenature||Denature||Annealing||Extension||cycle
 
|-
|-
-
|94&deg;C||98&deg;C||57&deg;C||68&deg;C||--
+
|1.0||2.5||2.5||1.5||1.5||0.5||15.5||25.0
-
|-
+
-
|2m||10s||30s||2m||30cycles
+
-
|}
+
-
</div>
+
-
<!-- ここまでをコピーしてね -->
+
-
<!-- ここから -->
+
-
<div class="experiment">
+
-
{| class="wikitable"
+
-
!Kai(400pg)||10x buffer KOD plus ver2||2M dNTP||MgSO4||Primer Mix(F&R)||Kod-plus ver2||MilliQ||total
+
|-
|-
|2.0||2.5||2.5||1.5||1.5||0.5||14.5||25.0
|2.0||2.5||2.5||1.5||1.5||0.5||14.5||25.0
-
|}
 
-
{| class="wikitable"
 
-
!PreDenature||Denature||Annealing||Extension||cycle
 
-
|-
 
-
|94&deg;C||98&deg;C||57&deg;C||68&deg;C||--
 
-
|-
 
-
|2m||10s||30s||2m||30cycles
 
-
|}
 
-
</div>
 
-
<!-- ここまでをコピーしてね -->
 
-
<!-- ここから -->
 
-
<div class="experiment">
 
-
{| class="wikitable"
 
-
!Kai(800pg)||10x buffer KOD plus ver2||2M dNTP||MgSO4||Primer Mix(F&R)||Kod-plus ver2||MilliQ||total
 
|-
|-
|4.0||2.5||2.5||1.5||1.5||0.5||12.5||25.0
|4.0||2.5||2.5||1.5||1.5||0.5||12.5||25.0
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|-
|-
|0.3||2.5||2.5||1.5||1.5||0.5||16.2||25.0
|0.3||2.5||2.5||1.5||1.5||0.5||16.2||25.0
-
|}
 
-
{| class="wikitable"
 
-
!PreDenature||Denature||Annealing||Extension||cycle
 
-
|-
 
-
|94&deg;C||98&deg;C||57&deg;C||68&deg;C||--
 
-
|-
 
-
|2m||10s||30s||2m||30cycles
 
-
|}
 
-
</div>
 
-
<!-- ここまでをコピーしてね -->
 
-
<!-- ここから -->
 
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<div class="experiment">
 
-
{| class="wikitable"
 
-
!Plac(200pg)||10x buffer KOD plus ver2||2M dNTP||MgSO4||Primer Mix(F&R)||Kod-plus ver2||MilliQ||total
 
|-
|-
|0.6||2.5||2.5||1.5||1.5||0.5||15.9||25.0
|0.6||2.5||2.5||1.5||1.5||0.5||15.9||25.0
-
|}
 
-
{| class="wikitable"
 
-
!PreDenature||Denature||Annealing||Extension||cycle
 
-
|-
 
-
|94&deg;C||98&deg;C||57&deg;C||68&deg;C||--
 
-
|-
 
-
|2m||10s||30s||2m||30cycles
 
-
|}
 
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</div>
 
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<!-- ここまでをコピーしてね -->
 
-
<!-- ここから -->
 
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<div class="experiment">
 
-
{| class="wikitable"
 
-
!Plac(400pg)||10x buffer KOD plus ver2||2M dNTP||MgSO4||Primer Mix(F&R)||Kod-plus ver2||MilliQ||total
 
|-
|-
|1.2||2.5||2.5||1.5||1.5||0.5||15.3||25.0
|1.2||2.5||2.5||1.5||1.5||0.5||15.3||25.0
-
|}
 
-
{| class="wikitable"
 
-
!PreDenature||Denature||Annealing||Extension||cycle
 
-
|-
 
-
|94&deg;C||98&deg;C||57&deg;C||68&deg;C||--
 
-
|-
 
-
|2m||10s||30s||2m||30cycles
 
-
|}
 
-
</div>
 
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<!-- ここまでをコピーしてね -->
 
-
<!-- ここから -->
 
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<div class="experiment">
 
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{| class="wikitable"
 
-
!Plac(800pg)||10x buffer KOD plus ver2||2M dNTP||MgSO4||Primer Mix(F&R)||Kod-plus ver2||MilliQ||total
 
|-
|-
|2.4||2.5||2.5||1.5||1.5||0.5||14.1||25.0
|2.4||2.5||2.5||1.5||1.5||0.5||14.1||25.0
-
|}
 
-
{| class="wikitable"
 
-
!PreDenature||Denature||Annealing||Extension||cycle
 
-
|-
 
-
|94&deg;C||98&deg;C||57&deg;C||68&deg;C||--
 
-
|-
 
-
|2m||10s||30s||2m||30cycles
 
-
|}
 
-
</div>
 
-
<!-- ここまでをコピーしてね -->
 
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<!-- ここから -->
 
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<div class="experiment">
 
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{| class="wikitable"
 
-
!Plac(nega)||10x buffer KOD plus ver2||2M dNTP||MgSO4||Primer Mix(F&R)||Kod-plus ver2||MilliQ||total
 
|-
|-
|0.0||2.5||2.5||1.5||1.5||0.5||16.5||25.0
|0.0||2.5||2.5||1.5||1.5||0.5||16.5||25.0
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</div>
</div>
<!-- ここまでをコピーしてね -->
<!-- ここまでをコピーしてね -->
 +
===Liquid Culture===
===Liquid Culture===
<!-- ここから -->
<!-- ここから -->

Revision as of 10:00, 24 September 2013

Contents

Sep 9

PCR

Nakagawa

KaiA(plasmid)10x bufferdNTPsMgSO4Primer(661-RBS-KaiA-fwd)Primer(kaiA-662-rev-ver2)Kod-plus ver2MilliQtotal
1.02.52.51.50.750.750.515.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C58.5°C68°C--
2min10s30s30s30cycles
KaiB(plasmid)10x bufferdNTPsMgSO4Primer(661-RBS-KaiA-fwd-ver2)Primer(kaiB-663-rev-ver2)Kod-plus ver2MilliQtotal
1.02.52.51.50.750.750.515.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C58.5°C68°C--
2min10s30s30s30cycles
P-KaiBC(PCR products)10x bufferdNTPsMgSO4Primer(prefix-P-kaiBC-fwd-ver2)Primer(P-KaiBC-suffix-rev-verw)Kod-plus ver2MilliQtotal
0.72.52.51.50.750.750.515.825
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C58.5°C68°C--
2min10s30s30s30cycles
DT(plasmid)10x bufferdNTPsMgSO4Primer(664-DT-fwd-ver2)Primer(DT-suffix-660-rev-ver2)Kod-plus ver2MilliQtotal
1.02.52.51.50.750.750.515.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C58.5°C68°C--
2min10s30s30s30cycles

Gel Extraction

Yoshida

LaneDNAEnzyme
1100bp ladder--
2KaiA--
3
4KaiB--
5
6P-KaiBC--
7
8DT--
9
10NC--
11100bp ladder--

File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx

Nameconcentration[µg/mL]260/280260/230
（´ω｀）
（ΦωΦ^）

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
9/8 pSB1C383.21.450.76
9/8 RBS-lysis2-DT72.81.670.97
9/8 spinach-DT152.61.881.97
Nuclease-Free water-0.4-14.300.25

Restriction Enzyme Digestion

じっけんしゃ

DNArestricion enzymerestriction enzymeBufferBSAMilliQtotal
2 cuts（°°） µL µL µL µL µL µL µL
NC µL µL µL µL µL µL µL

Transformation

じっけんしゃ

NameSampleCompetent CellsTotalPlate
いでんしなんちゃら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

Ligation

No Name

stateVectorInserterLigation High ver.2
experiment9/8 F1-attenuator(XbaI&PstI)5.3µg9/8 pSB1C3(XbaI&PstI)8.9µg3.5µg
experiment9/8 F3m2-attenuator(XbaI&PstI)4.5µg9/8 pSB1C3(XbaI&PstI)8.9µg3.5µg
experiment9/8 F1-antisense(XbaI&PstI)1.7µg9/8 pSB1C3(XbaI&PstI)8.9µg3.5µg
experiment9/8 F6-antisense(XbaI&PstI)2.9µg9/8 pSB1C3(XbaI&PstI)8.9µg3.5µg
experiment9/8 aptamer12-P(EcoRI&SpeI)3.1µg9/8 pSB1C3(EcoRI&SpeI)8.8µg3.5µg
experiment9/8 aptamer12-1M(EcoRI&SpeI)3.1µg9/8 pSB1C(EcoRI&SpeI)38.8µg3.5µg

Fe(Ⅲ)

Shimizu & Hirano

Colony PCR

No name

Samplebase pair
9/8 Plac-spinach-DT-(1)659
9/8 Plac-spinach-DT-(2)659
9/8 Pcon-pt181atte-DT-(1)584
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5m30s30s40s30cycles

Gel Extraction

じっけんしゃ

LaneDNAEnzyme
1100bp ladder--
2Spinach-DTXbaI+PstI
3
5RBS-lacZα-DTXbaI+PstI
6

File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx

Namequantityconcentration[µg/mL]260/280260/230
（´ω｀）
（ΦωΦ^）

Ligation

じっけんしゃ

stateVectorInserterLigation High ver.2
experimentnamexx µg/mLnamexx µg/mLxx µg/mL
NCnamexx µg/mLnamexx µg/mLxx µg/mL

PCR

Tatsui and Yoshida

KaiC10x buffer KOD plus ver22M dNTPMgSO4Primer Mix(F&R)Kod-plus ver2MilliQtotal
0.52.52.51.51.50.516.025.0
1.02.52.51.51.50.515.525.0
2.02.52.51.51.50.514.525.0
4.02.52.51.51.50.512.525.0
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2m10s30s2m30cycles
Plac(100pg)10x buffer KOD plus ver22M dNTPMgSO4Primer Mix(F&R)Kod-plus ver2MilliQtotal
0.32.52.51.51.50.516.225.0
0.62.52.51.51.50.515.925.0
1.22.52.51.51.50.515.325.0
2.42.52.51.51.50.514.125.0
0.02.52.51.51.50.516.525.0
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2m10s30s2m30cycles

Liquid Culture

No name

Samplemedium

Restriction Enzyme Digestion

じっけんしゃ

DNArestricion enzymerestriction enzymeBufferBSAMilliQtotal
2 cuts（°°） µL µL µL µL µL µL µL
NC µL µL µL µL µL µL µL

PCR

Yoshida

KaiA(KaiABC) 100pg/µL10x buffer KOD plus ver2dNTPsMgSO4Primer Mix(Fwd&Rev)KOD plus ver2MilliQtotal
2.02.52.51.51.50.514.525
4.02.52.51.51.50.512.525
8.02.52.51.51.50.58.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C60°C68°C--
2m10s30s1m30cycles
PKaiBC 400pg/µL10x buffer KOD plus ver2dNTPsMgSO4Primer Mix(Fwd&Rev)KOD plus ver2MilliQtotal
0.52.52.51.51.50.516.025
1.02.52.51.51.50.515.525
2.02.52.51.51.50.514.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C60°C68°C--
2m10s30s1m30cycles

Liquid Culture

No name

Samplemedium
9/2 Spinach-DTPlusgrow medium(+CP)
8/29 Plux-RBS-GFP-DTPlusgrow medium
9/6 master plate Plusgrow medium(+Amp)
3 Pcon-PT181 attenuatorPlusgrow medium

Gel Extraction

No name

LaneDNAEnzyme
11kbp ladder--
3lysis1XbaI+PstI
4
5
6

File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx

Nameconcentration[µg/mL]260/280260/230
（´ω｀）
（ΦωΦ^）

No name

LaneDNAEnzyme
11kbp ladder--
3lysis3XbaI+PstI
4
5
6

File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx

Nameconcentration[µg/mL]260/280260/230
（´ω｀）
（ΦωΦ^）

Electrophoresis

Yoshida

LaneSampleEnzyme1Enzyme2
11kb ladder----
2Plac 100pg----
3Plac 200pg----
4Plac 400pg----
5Plac 800pg----
6NC----
71kb ladder----

File:Igku xxxxxx.xxx

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