Template:Kyoto/Notebook/Sep 9

From 2013.igem.org

(Difference between revisions)
(→Gel Extraction)
(→Restriction Enzyme Digestion)
 
(62 intermediate revisions not shown)
Line 52: Line 52:
|}
|}
</div>
</div>
 +
===Gel Extraction===
 +
<!-- こっから -->
 +
<div class="experiment">
 +
<span class="author">Yoshida</span>
 +
{| class="wikitable"
 +
!Lane||DNA||Enzyme
 +
|-
 +
|1||100bp ladder||--
 +
|-
 +
|2||rowspan="2"|KaiA||rowspan="2"|--
 +
|-
 +
|3
 +
|-
 +
|4||rowspan="2"|KaiB||rowspan="2"|--
 +
|-
 +
|5
 +
|-
 +
|6||rowspan="2"|P-KaiBC||rowspan="2"|--
 +
|-
 +
|7
 +
|-
 +
|8||rowspan="2"|DT||rowspan="2"|--
 +
|-
 +
|9
 +
|-
 +
|10||NC||--
 +
|-
 +
|11||100bp ladder||--
 +
|}
 +
[[File:igku_992.jpg]]<br>
 +
[[File:igku_993.jpg]]<br>
 +
{| class="wikitable"
 +
!Name||concentration[&micro;g/mL]||260/280||260/230
 +
|-
 +
|KaiC||28.5||1.26||11.92
 +
|-
 +
|Plac||53.9||1.82||1.61
 +
|-
 +
|KaiB||59.6||1.61||2.20
 +
|-
 +
|KaiB||76.5||1.86||1.44
 +
|-
 +
|DT||98.7||1.47||1.43
 +
|}
 +
</div>
 +
<!-- ここまでをコピーしてね -->
 +
===Miniprep===
===Miniprep===
<div class="experiment">
<div class="experiment">
Line 67: Line 114:
|}
|}
</div>
</div>
 +
===Restriction Enzyme Digestion===
===Restriction Enzyme Digestion===
-
<!-- こっから -->
 
<div class="experiment">
<div class="experiment">
-
<span class="author">じっけんしゃ</span>
+
<span class="author">No name</span>
{| class="wikitable"
{| class="wikitable"
-
! ||DNA||restricion enzyme||restriction enzyme||Buffer||BSA||MilliQ||total
+
! ||9/9 pSB1C3||XbaI||PstI||Buffer||BSA||MilliQ||total
|-
|-
-
|2 cuts||（°°） &micro;L|| &micro;L|| &micro;L|| &micro;L|| &micro;L|| &micro;L|| &micro;L
+
|2 cuts||24&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||22&micro;L||30&micro;L*
|-
|-
-
|NC|| &micro;L|| &micro;L|| &micro;L|| &micro;L|| &micro;L|| &micro;L|| &micro;L
+
|NC||1.2&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||6.8 &micro;L||10&micro;L
|}
|}
</div>
</div>
-
<!-- ここまでをコピーしてね -->
+
<div class="experiment">
 +
{| class="wikitable"
 +
! ||9/9 pSB1C3||XbaI||PstI||Buffer||BSA||MilliQ||total
 +
|-
 +
|2 cuts||27.4&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||22&micro;L||30&micro;L*
 +
|-
 +
|NC||1.3&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||6.7&micro;L||10&micro;L
 +
|}
 +
</div>
 +
<div class="experiment">
 +
{| class="wikitable"
 +
! ||9/9 spinach-DT||XbaI||PstI||Buffer||BSA||MilliQ||total
 +
|-
 +
|2 cuts||10.8&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||22&micro;L||30&micro;L*
 +
|-
 +
|NC||0.5&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||7.5&micro;L||10&micro;L
 +
|}
 +
</div>
 +
*"*"=be evaporated
 +
 
===Transformation===
===Transformation===
-
<!-- こっから -->
 
<div class="experiment">
<div class="experiment">
-
<span class="author">じっけんしゃ</span>
+
<span class="author">No name</span>
{| class="wikitable"
{| class="wikitable"
!Name||Sample||Competent Cells||Total||Plate
!Name||Sample||Competent Cells||Total||Plate
|-
|-
-
|いでんし||なんちゃら&micro;L||ごにょごにょ&micro;L||ほにゃらら&micro;L||こーせーぶっしつ
+
|Pcon+RBS-luxI-DT||2&micro;L||20&micro;L||22&micro;L||Amp
|-
|-
-
|いかどうよう|| || || ||
+
|Pcon-RBS-luxR-DT+Plux-GFP||2&micro;L||20&micro;L||22&micro;L||
|-
|-
-
| || || || ||
+
|Ptet-PT181 antisense+spinach-DT-Pbad/araC+RBS-lux-DT||2&micro;L||20&micro;L||22&micro;L||
|}
|}
</div>
</div>
-
<!-- ここまでをコピーしてね -->
+
 
===Ligation===
===Ligation===
<div class="experiment">
<div class="experiment">
Line 101: Line 166:
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
|-
|-
-
|experiment||name||xx &micro;g/mL||name||xx &micro;g/mL||xx &micro;g/mL
+
|experiment||9/8 F1-attenuator(XbaI&PstI)||5.3&micro;g||9/8 pSB1C3(XbaI&PstI)||8.9&micro;g||3.5&micro;g
|-
|-
-
|NC||name||xx &micro;g/mL||name||xx &micro;g/mL||xx &micro;g/mL
+
|experiment||9/8 F3m2-attenuator(XbaI&PstI)||4.5&micro;g||9/8 pSB1C3(XbaI&PstI)||8.9&micro;g||3.5&micro;g
 +
|-
 +
|experiment||9/8 F1-antisense(XbaI&PstI)||1.7&micro;g||9/8 pSB1C3(XbaI&PstI)||8.9&micro;g||3.5&micro;g
 +
|-
 +
|experiment||9/8 F6-antisense(XbaI&PstI)||2.9&micro;g||9/8 pSB1C3(XbaI&PstI)||8.9&micro;g||3.5&micro;g
 +
|-
 +
|experiment||9/8 aptamer12-P(EcoRI&SpeI)||3.1&micro;g||9/8 pSB1C3(EcoRI&SpeI)||8.8&micro;g||3.5&micro;g
 +
|-
 +
|experiment||9/8 aptamer12-1M(EcoRI&SpeI)||3.1&micro;g||9/8 pSB1C(EcoRI&SpeI)3||8.8&micro;g||3.5&micro;g
|}
|}
</div>
</div>
 +
 +
===M9 medium===
 +
<div class="experiment">
 +
<span class="author">Shimizu & Hirano</span>
 +
{| class="wikitable"
 +
!EDTA consenlation||Na2HPO4||KH2PO4||NaCl||NH4Cl||Fe(&#8546;)-EDTA||distilled water||total
 +
|-
 +
|1mMol||300mg||150mg||25mg||50mg||21mg||up to 10mL||10mL
 +
|-
 +
|10mMol||300mg||150mg||25mg||50mg||210.5mg||up to 10mL||10mL
 +
|-
 +
|50mMol||300mg||150mg||25mg||50mg||1052.7mg||up to 10mL||10mL
 +
|}
 +
</div>
 +
===Colony PCR===
===Colony PCR===
<div class="experiment">
<div class="experiment">
Line 113: Line 201:
|-
|-
|9/8 Plac-spinach-DT-(1)||659
|9/8 Plac-spinach-DT-(1)||659
 +
|-
 +
|9/8 Plac-spinach-DT-(2)||659
 +
|-
 +
|9/8 Pcon-pt181attenuator-DT-(1)||584
|}
|}
{| class="wikitable"
{| class="wikitable"
Line 119: Line 211:
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||--
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||--
|-
|-
-
|5m||30s||30s||40s||30cycles
+
|5min||30s||30s||40s||30cycles
|}
|}
</div>
</div>
 +
 +
===Gel Extraction===
<div class="experiment">
<div class="experiment">
 +
<span class="author">No name</span>
{| class="wikitable"
{| class="wikitable"
-
!Sample||base pair
+
!Lane||DNA||Enzyme
|-
|-
-
|9/8 Plac-spinach-DT-(2)||659
+
|1||100bp ladder||--
 +
|--
 +
|2||rowspan="2"|Spinach-DT||rowspan="2"|XbaI+PstI
 +
|-
 +
|3
 +
|-
 +
|5||rowspan="2"|RBS-lacZ&alpha;-DT||rowspan="2"|XbaI+PstI
 +
|-
 +
|6
|}
|}
 +
[[File:igku_995.jpg]]<br>
 +
 +
</div>
 +
<div class="experiment">
{| class="wikitable"
{| class="wikitable"
-
!PreDenature||Denature||Annealing||Extension||cycle
+
!Lane||DNA||Enzyme
|-
|-
-
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||--
+
|1||100bp ladder||--
 +
|--
 +
|3||rowspan="2"|pSB1C3||rowspan="2"|XbaI+PstI
|-
|-
-
|5m||30s||30s||40s||30cycles
+
|4
|}
|}
 +
[[File:igku_996.jpg]]<br>
 +
</div>
</div>
 +
 +
===Ligation===
<div class="experiment">
<div class="experiment">
 +
<span class="author">No name</span>
{| class="wikitable"
{| class="wikitable"
-
!Sample||base pair
+
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
 +
|-
 +
|experiment||pSB1C3(XbaI+PstI)||xx &micro;L||9/8 F1-attenuator(XbaI+PstI)||xx &micro;L||xx &micro;L
 +
|-
 +
|experiment||pSB1C3(XbaI+PstI)||xx &micro;L||9/8 F3m2-attenuator(XbaI+PstI)||xx &micro;L||xx &micro;L
 +
|}
 +
</div>
 +
 
 +
===PCR===
 +
<div class="experiment">
 +
<span class="author">Tatsui and Yoshida</span>
 +
{| class="wikitable"
 +
!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
 +
|-
 +
|1.0||2.5||2.5||1.5||1.5||0.5||15.5||25.0
 +
|-
 +
|2.0||2.5||2.5||1.5||1.5||0.5||14.5||25.0
|-
|-
-
|9/8 Pcon-pt181atte-DT-(1)||584
+
|4.0||2.5||2.5||1.5||1.5||0.5||12.5||25.0
|}
|}
{| class="wikitable"
{| class="wikitable"
!PreDenature||Denature||Annealing||Extension||cycle
!PreDenature||Denature||Annealing||Extension||cycle
|-
|-
-
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||--
+
|94&deg;C||98&deg;C||57&deg;C||68&deg;C||--
|-
|-
-
|5m||30s||30s||40s||30cycles
+
|2min||10s||30s||2min||30cycles
|}
|}
</div>
</div>
-
===Gel Extraction===
 
<div class="experiment">
<div class="experiment">
-
<span class="author">じっけんしゃ</span>
 
{| class="wikitable"
{| class="wikitable"
-
!Lane||DNA||Enzyme
+
!Plac(100pg)||10x buffer KOD plus ver2||2M dNTP||MgSO4||Primer Mix(F&R)||Kod-plus ver2||MilliQ||total
|-
|-
-
|1||100bpladder||--
+
|0.3||2.5||2.5||1.5||1.5||0.5||16.2||25.0
-
|--
+
-
|2||rowspan="2"|Spinach-DT||rowspan="2"|X+P
+
|-
|-
-
|3
+
|0.6||2.5||2.5||1.5||1.5||0.5||15.9||25.0
|-
|-
-
|5||rowspan="2"|RBS-lacZ&alpha;-DT||rowspan="2"|X+P
+
|1.2||2.5||2.5||1.5||1.5||0.5||15.3||25.0
|-
|-
-
|6
+
|2.4||2.5||2.5||1.5||1.5||0.5||14.1||25.0
 +
|-
 +
|0.0||2.5||2.5||1.5||1.5||0.5||16.5||25.0
|}
|}
-
[[File:igku_xxbeforexx.xxx]]<br>
 
-
[[File:igku_xxafterxx.xxx]]<br>
 
{| class="wikitable"
{| class="wikitable"
-
!Name||quantity||concentration[&micro;g/mL]||260/280||260/230
+
!PreDenature||Denature||Annealing||Extension||cycle
|-
|-
-
|（´ω｀）|| || || ||
+
|94&deg;C||98&deg;C||57&deg;C||68&deg;C||--
|-
|-
-
|（ΦωΦ^）|| || || ||
+
|2min||10s||30s||2min||30cycles
|}
|}
</div>
</div>
-
===Ligation===
+
===Liquid Culture===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Sample||medium
 +
|-
 +
|pSB2C3(master)-2||Plusgrow medium(+CP)
 +
|}
 +
</div>
 +
===Restriction Enzyme Digestion===
<div class="experiment">
<div class="experiment">
-
<span class="author">じっけんしゃ</span>
+
<span class="author">No name</span>
{| class="wikitable"
{| class="wikitable"
-
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
+
! ||8/17 lysis1-2||XbaI||PstI||BSA||Buffer||MilliQ||total
 +
|-
 +
|2 cuts||8.1&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||13.9&micro;L||30&micro;L
 +
|-
 +
|NC||0.4&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||7.6&micro;L||10&micro;L
 +
|}
 +
{| class="wikitable"
 +
! ||8/17 lysis3-1||XbaI||PstI||BSA||Buffer||MilliQ||total
 +
|-
 +
|2 cuts||6.1&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||15.9&micro;L||30&micro;L
 +
|-
 +
|NC||0.3&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||7.7&micro;L||10&micro;L
 +
|}
 +
{| class="wikitable"
 +
! ||9/15 Plac||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
-
|experiment||name||xx &micro;g/mL||name||xx &micro;g/mL||xx &micro;g/mL
+
|1 cuts||12.1&micro;L||0&micro;L||1&micro;L||3&micro;L||3&micro;L||10.9&micro;L||30&micro;L
|-
|-
-
|NC||name||xx &micro;g/mL||name||xx &micro;g/mL||xx &micro;g/mL
+
|NC||0.3&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||7.7&micro;L||10&micro;L
|}
|}
</div>
</div>
Line 193: Line 344:
<!-- ここから -->
<!-- ここから -->
<div class="experiment">
<div class="experiment">
-
<span class="author">Tatsui and Yoshida</span>
+
<span class="author">Yoshida</span>
{| class="wikitable"
{| class="wikitable"
-
!genome DNA||KOD plus||10x buffer||dNTP||MgSO4||SasA_fwd primer||SasA_rev primer||MilliQ||total
+
!KaiA(KaiABC) 100pg/&micro;L||10x buffer KOD plus ver2||dNTPs||MgSO4||Primer Mix(Fwd&Rev)||KOD plus ver2||MilliQ||total
|-
|-
-
|KaiC(100pg)||0.5||2.5||2.5||1.5||0.75||0.75|| ||25
+
|2.0||2.5||2.5||1.5||1.5||0.5||14.5||25
 +
|-
 +
|4.0||2.5||2.5||1.5||1.5||0.5||12.5||25
 +
|-
 +
|8.0||2.5||2.5||1.5||1.5||0.5||8.5||25
|}
|}
{| class="wikitable"
{| class="wikitable"
!PreDenature||Denature||Annealing||Extension||cycle
!PreDenature||Denature||Annealing||Extension||cycle
|-
|-
-
|　&deg;C||　&deg;C||　&deg;C||　&deg;C||--
+
|94&deg;C||98&deg;C||60&deg;C||68&deg;C||--
|-
|-
-
|（◎Д◎）　||　||　||　||
+
|2min||10s||30s||1min||30cycles
|}
|}
</div>
</div>
-
<!-- ここまでをコピーしてね -->
+
<div class="experiment">
 +
{| class="wikitable"
 +
!PKaiBC 400pg/&micro;L||10x buffer KOD plus ver2||dNTPs||MgSO4||Primer Mix(Fwd&Rev)||KOD plus ver2||MilliQ||total
 +
|-
 +
|0.5||2.5||2.5||1.5||1.5||0.5||16.0||25
 +
|-
 +
|1.0||2.5||2.5||1.5||1.5||0.5||15.5||25
 +
|-
 +
|2.0||2.5||2.5||1.5||1.5||0.5||14.5||25
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||98&deg;C||60&deg;C||68&deg;C||--
 +
|-
 +
|2m||10s||30s||1m||30cycles
 +
|}
 +
</div>
 +
===Liquid Culture===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Sample||medium
 +
|-
 +
|9/2 Spinach-DT||Plusgrow medium(+CP)
 +
|-
 +
|8/29 Plux-RBS-GFP-DT||Plusgrow medium
 +
|-
 +
|9/6 master plate ||Plusgrow medium(+Amp)
 +
|-
 +
|3 Pcon-PT181 attenuator||Plusgrow medium
 +
|}
 +
</div>
 +
===Gel Extraction===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Lane||DNA||Enzyme
 +
|-
 +
|1||1kbp ladder||--
 +
|-
 +
|3||rowspan="4"|lysis1||rowspan="4"|XbaI+PstI
 +
|-
 +
|4
 +
|-
 +
|5
 +
|-
 +
|6
 +
|}
 +
[[File:igku_9910.jpg]]<br>
 +
[[File:igku_9911.jpg]]<br>
 +
</div>
 +
<div class="experiment">
 +
{| class="wikitable"
 +
!Lane||DNA||Enzyme
 +
|-
 +
|1||1kbp ladder||--
 +
|-
 +
|3||rowspan="4"|lysis3||rowspan="4"|XbaI+PstI
 +
|-
 +
|4
 +
|-
 +
|5
 +
|-
 +
|6
 +
|}
 +
[[File:igku_9912.jpg]]<br>
 +
[[File:igku_9913.jpg]]<br>
 +
{| class="wikitable"
 +
!Name||concentration[&micro;g/mL]||260/280||260/230
 +
|-
 +
|（´ω｀）|| || ||
 +
|-
 +
|（ΦωΦ^）|| || ||
 +
|}
 +
</div>
 +
 
 +
===Electrophoresis===
 +
<div class="experiment">
 +
<span class="author">Yoshida</span>
 +
{| class="wikitable"
 +
!Lane||Sample||Enzyme1||Enzyme2
 +
|-
 +
|1||1kb ladder||--||--
 +
|-
 +
|2||Plac 100pg||--||--
 +
|-
 +
|3||Plac 200pg||--||--
 +
|-
 +
|4||Plac 400pg||--||--
 +
|-
 +
|5||Plac 800pg||--||--
 +
|-
 +
|6||NC||--||--
 +
|-
 +
|7||1kbp ladder||--||--  
 +
|}
 +
[[File:igku_9914.jpg]]<br>
 +
</div>

Latest revision as of 20:10, 27 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--

Igku 992.jpg
Igku 993.jpg

Nameconcentration[µg/mL]260/280260/230
KaiC28.51.2611.92
Plac53.91.821.61
KaiB59.61.612.20
KaiB76.51.861.44
DT98.71.471.43

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

No name

9/9 pSB1C3XbaIPstIBufferBSAMilliQtotal
2 cuts24µL1µL1µL3µL3µL22µL30µL*
NC1.2µL0µL0µL1µL1µL6.8 µL10µL
9/9 pSB1C3XbaIPstIBufferBSAMilliQtotal
2 cuts27.4µL1µL1µL3µL3µL22µL30µL*
NC1.3µL0µL0µL1µL1µL6.7µL10µL
9/9 spinach-DTXbaIPstIBufferBSAMilliQtotal
2 cuts10.8µL1µL1µL3µL3µL22µL30µL*
NC0.5µL0µL0µL1µL1µL7.5µL10µL
  • "*"=be evaporated

Transformation

No name

NameSampleCompetent CellsTotalPlate
Pcon+RBS-luxI-DT2µL20µL22µLAmp
Pcon-RBS-luxR-DT+Plux-GFP2µL20µL22µL
Ptet-PT181 antisense+spinach-DT-Pbad/araC+RBS-lux-DT2µL20µL22µL

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

M9 medium

Shimizu & Hirano

EDTA consenlationNa2HPO4KH2PO4NaClNH4ClFe(Ⅲ)-EDTAdistilled watertotal
1mMol300mg150mg25mg50mg21mgup to 10mL10mL
10mMol300mg150mg25mg50mg210.5mgup to 10mL10mL
50mMol300mg150mg25mg50mg1052.7mgup to 10mL10mL

Colony PCR

No name

Samplebase pair
9/8 Plac-spinach-DT-(1)659
9/8 Plac-spinach-DT-(2)659
9/8 Pcon-pt181attenuator-DT-(1)584
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s40s30cycles

Gel Extraction

No name

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

Igku 995.jpg

LaneDNAEnzyme
1100bp ladder--
3pSB1C3XbaI+PstI
4

Igku 996.jpg

Ligation

No name

stateVectorInserterLigation High ver.2
experimentpSB1C3(XbaI+PstI)xx µL9/8 F1-attenuator(XbaI+PstI)xx µLxx µL
experimentpSB1C3(XbaI+PstI)xx µL9/8 F3m2-attenuator(XbaI+PstI)xx µLxx µL

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--
2min10s30s2min30cycles
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--
2min10s30s2min30cycles

Liquid Culture

No name

Samplemedium
pSB2C3(master)-2Plusgrow medium(+CP)

Restriction Enzyme Digestion

No name

8/17 lysis1-2XbaIPstIBSABufferMilliQtotal
2 cuts8.1µL1µL1µL3µL3µL13.9µL30µL
NC0.4µL0µL0µL1µL1µL7.6µL10µL
8/17 lysis3-1XbaIPstIBSABufferMilliQtotal
2 cuts6.1µL1µL1µL3µL3µL15.9µL30µL
NC0.3µL0µL0µL1µL1µL7.7µL10µL
9/15 PlacXbaIPstIBSABufferMilliQtotal
1 cuts12.1µL0µL1µL3µL3µL10.9µL30µL
NC0.3µL0µL0µL1µL1µL7.7µL10µL

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--
2min10s30s1min30cycles
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

Igku 9910.jpg
Igku 9911.jpg

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

Igku 9912.jpg
Igku 9913.jpg

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

Electrophoresis

Yoshida

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

Igku 9914.jpg

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