Template:Kyoto/Notebook/Sep 4

From 2013.igem.org

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(→Gel Extraction)
(→Electrophoresis)
 
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Line 1: Line 1:
==Sep 4==
==Sep 4==
 +
===Ligation===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
 +
|-
 +
|experiment||9/3 DT(EcoRI&XbaI)||3.2&micro;L||9/3 RBS-lysis1||2.4&micro;L||2.7&micro;L
 +
|-
 +
|experiment||9/3 DT(EcoRI&XbaI) ||3.0&micro;L ||9/3 RBS-lysis2||4.7&micro;L ||4.0&micro;L
 +
|-
 +
|experiment||9/3 DT(EcoRI&XbaI) ||3.0&micro;L ||9/3 RBS-lysis3||8.8&micro;L ||4.0&micro;L
 +
|-
 +
|experiment||9/3 DT(EcoRI&XbaI) ||3.0&micro;L ||9/3 aptamer12_1R(EcoRI&SpeI) ||1.9&micro;L ||2.45&micro;L
 +
|-
 +
|experiment||9/2 pSB1C3(XbaI&PstI)||10.6&micro;L ||9/3 pT181attenuator (XbaI& PstI)||3.2&micro;L ||4.0&micro;L
 +
|-
 +
|experiment||9/2 pSB1C3(XbaI&PstI)||9.6&micro;L ||9/3 pT181antisense (XbaI& PstI)||2.1&micro;L ||4.0&micro;L
 +
|-
 +
|experiment||9/2 pSB1C3(EcoRI&SpeI)||16.7&micro;L ||9/3 aptamer12_1R(EcoRI&SpeI) ||2.0&micro;L ||4.0&micro;L
 +
|}
 +
incubate 16 &deg;C 1 hour
 +
</div>
 +
===Transformation===
 +
<div class="experiment">
 +
<span class="author">Tatsui</span>
 +
{| class="wikitable"
 +
!Name||Sample||Competent Cells||Plate
 +
|-
 +
|9/4 RBS-lysis1-DT ||2&micro;L||20&micro;L||Amp
 +
|-
 +
|9/4 RBS-lysis2-DT ||2&micro;L||20&micro;L||Amp
 +
|-
 +
|9/4 RBS-lysis3-DT ||2&micro;L||20&micro;L||Amp
 +
|-
 +
|9/4 aptamer12_1R-DT||2&micro;L||20&micro;L||Amp
 +
|-
 +
|9/4 pT181attenuator –pSB1C3||2&micro;L||20&micro;L||Amp
 +
|-
 +
|9/4 pT181antisense-pSB1C3 ||2&micro;L||20&micro;L||Amp
 +
|-
 +
|9/4 aptamer12_1R –pSB1C3||2&micro;L||20&micro;L||Amp
 +
|-
 +
|9/4 pSB4K5(2013plate5 56) ||2&micro;L||20&micro;L||Amp
 +
|-
 +
|9/3 Mutagenesis product 1(KaiABC) ||2&micro;L||20&micro;L||Amp
 +
|-
 +
|9/3 Mutagenesis product 2(NC) ||2&micro;L||20&micro;L||Amp
 +
|}
 +
</div>
-
===Gel Extraction===
+
===Miniprep===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{|class="wikitable"
 +
!DNA||concentration[&micro;g/mL]||260/280||260/230
 +
|-
 +
|DT||193.1||1.93||1.81
 +
|-
 +
|Spinach-DT||373.3||1.92||1.69
 +
|-
 +
|RBS-lysis1||332.0||1.95||1.62
 +
|}
 +
===Master Plate===
 +
<!-- ここから -->
<div class="experiment">
<div class="experiment">
-
<span class="author">No name</span></div>
+
<span class="author">Hirano</span>
 +
{| class="wikitable"
 +
!Number||Use LB plate (+CP)
 +
|-
 +
|1||8/10 Plac(CP)-2
 +
|-
 +
|2|| 8/10 Plac(CP)-2
 +
|-
 +
|3||8/10 Plac(CP)-2
 +
|}
 +
{| class="wikitable"
 +
!Number||Use LB plate (+Amp)
 +
|-
 +
|1||8/9 J23100(Amp)-1
 +
|-
 +
|2||8/9 J23100(Amp)-1
 +
|-
 +
|3||8/9 J23100(Amp)-1
 +
|}
 +
</div>
-
1
+
===Liquid Culture===
 +
<div class="experiment">
 +
<span class="author">Hirano</span>
 +
{| class="wikitable"
 +
!Sample||medium
 +
|-
 +
|8/10 Plac(CP)-2||Plusgrow medium(+CP)
 +
|-
 +
|8/9 J23100(Amp)-1||Plusgrow medium(+Amp)
 +
|}
 +
</div>
 +
===PCR===
 +
<div class="experiment">
 +
<span class="author">Hirano</span>
{| class="wikitable"
{| class="wikitable"
-
!Lane||DNA||Enzyme
+
!8/29 P&lambda;-RBS-luxI-DT||KOD plus||10x buffer||dNTP||MgSO4||F primer (RBS-luxI-DT-cloning-fwd) || R primer(RBS-luxI-DT-cloning-rev)||MilliQ||total
|-
|-
-
|1||100bp ladder||--
+
|0.3&micro;L||0.5&micro;L ||2.5&micro;L ||2.5&micro;L ||1.5&micro;L ||0.75&micro;L ||0.75&micro;L ||16.2&micro;L ||25&micro;L
 +
|}
 +
{| class="wikitable"
 +
!8/31 Pbad/araC-RBS-RFP(1)||KOD plus||10x buffer||dNTP||MgSO4||F primer (Pbad/araC-pSB1C3-cloning-fond) ||R primer(Pbad/araC-cloning-rex)||MilliQ||total
|-
|-
-
|2||Pcon||SpeI&PstI
+
|0.3&micro;L||0.5&micro;L ||2.5&micro;L ||2.5&micro;L ||1.5&micro;L ||0.75&micro;L ||0.75&micro;L ||16.2&micro;L ||25&micro;L
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
|-
|-
-
|3||Pcon||SpeI&PstI
+
|94&deg;C||98&deg;C||65&deg;C||68&deg;C||--
|-
|-
-
|4||--||--
+
|5min||10s||30s||3min10s||30cycles
 +
|}
 +
</div>
 +
 
 +
===Restriction Enzyme Digestion===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
! ||8/21 Pconst||SpeI||PstI||Buffer||BSA||MilliQ||total
|-
|-
-
|5||Plac||SpeI&PstI
+
|2 cuts||10.5&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||0.3&micro;L||14.2&micro;L||30 &micro;L
|-
|-
-
|6||Plac||SpeI&PstI
+
|NC||0.5&micro;L||0&micro;L||0&micro;||1.0&micro;L||0.1&micro;L||8.4&micro;L||10 &micro;L
 +
|}
 +
 
 +
{| class="wikitable"
 +
! ||8/29 Plac||SpeI||PstI||Buffer||BSA||MilliQ||total
 +
|-
 +
|2 cuts||13.0&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||0.3&micro;L||11.7&micro;L||30 &micro;L
 +
|-
 +
|NC||0.7&micro;L||0&micro;L||0&micro;||1.0&micro;L||0.1&micro;L|| 8.2&micro;L||10 &micro;L
 +
|}
 +
 +
{| class="wikitable"
 +
! ||8/20 Ptet||SpeI||PstI||Buffer||BSA||MilliQ||total
 +
|-
 +
|2 cuts||9.0&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||0.3&micro;L||15.7&micro;L||30 &micro;L
|-
|-
 +
|NC||0.5&micro;L||0&micro;L||0&micro;||1.0&micro;L||0.1&micro;L|| 8.4&micro;L||10 &micro;L
 +
|}
 +
{| class="wikitable"
 +
! ||8/20 RBS-tetR-DT||XbaI||PstI||Buffer||BSA||MilliQ||total
 +
|-
 +
|2 cuts||8.5&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||0.3&micro;L||16.2&micro;L||30 &micro;L
 +
|-
 +
|NC||0.5&micro;L||0&micro;L||0&micro;||1.0&micro;L||0.1&micro;L|| 8.4&micro;L||10 &micro;L
 +
|}
 +
{| class="wikitable"
 +
! ||spinach -DT||XbaI||PstI||Buffer||BSA||MilliQ||total
 +
|-
 +
|2 cuts||5.4&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||3.0&micro;L||16.6&micro;L||30 &micro;L
 +
|-
 +
|NC||0.3&micro;L||0&micro;L||0&micro;||1.0&micro;L||1.0&micro;L|| 7.7&micro;L||10 &micro;L
 +
|}
 +
{| class="wikitable"
 +
! ||8/17 RBS-lacZ&alpha;-DT ||XbaI||PstI||Buffer||BSA||MilliQ||total
 +
|-
 +
|2 cuts||11&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||3.0&micro;L||11&micro;L||30 &micro;L
 +
|-
 +
|NC||0.5&micro;L||0&micro;L||0&micro;||1.0&micro;L||1.0&micro;L||7.5&micro;L||10 &micro;L
|}
|}
-
[[File:igku_xxbeforexx.xxx]]<br>
+
</div>
-
[[File:igku_xxafterxx.xxx]]<br>
+
 
 +
===Transformation===
 +
<div class="experiment">
 +
<span class="author">No name</span>
{| class="wikitable"
{| class="wikitable"
-
!Name||quantity||concentration[&micro;g/mL]||260/280||260/230
+
!Name||Sample||Competent Cells||Total||Plate
|-
|-
-
|（´ω｀）|| || || ||
+
|Ptrc KaiC||1&micro;L||10&micro;L||11&micro;L||Amp
|-
|-
-
|（ΦωΦ^）|| || || ||
+
|Ptrc KaiB||1&micro;L||10&micro;L||11&micro;L||Amp
 +
|-
 +
|pS&Omega; ||1&micro;L||10&micro;L||11&micro;L||Amp
|}
|}
</div>
</div>
 +
===Electrophoresis===
<div class="experiment">
<div class="experiment">
-
<span class="author">No name</span></div>
+
<span class="author">No name</span><br>
 +
1
 +
 
 +
{| class="wikitable"
 +
!Lane||Sample||Enzyme1||Enzyme2
 +
|-
 +
|1||100bp ladder||--||--
 +
|-
 +
|2||Pconst||SpeI||PstI
 +
|-
 +
|3||Pconst NC||--||--
 +
|-
 +
|4||Plac||SpeI||PstI
 +
|-
 +
|5||Plac NC||--||--
 +
|-
 +
|6||100bp ladder||--||--
 +
|}
2
2
 +
{| class="wikitable"
 +
!Lane||Sample||Enzyme1||Enzyme2
 +
|-
 +
|1||100bp ladder||--||--
 +
|-
 +
|2||Ptet||SpeI||PstI
 +
|-
 +
|3||Ptet NC||--||--
 +
|-
 +
|4||RBS-tetR-DT||XbaI||PstI
 +
|-
 +
|5||RBS-tetR-DT NC||--||--
 +
|-
 +
|6||100bp ladder||--||--
 +
|}
 +
3
 +
{| class="wikitable"
 +
!Lane||Sample||Enzyme1||Enzyme2
 +
|-
 +
|1||100bp ladder||--||--
 +
|-
 +
|2||Spinach-DT||XbaI||PstI
 +
|-
 +
|3||Spinach-DT NC||--||--
 +
|-
 +
|4||RBS-lacZ&alpha;-DT||XbaI||PstI
 +
|-
 +
|5||RBS-lac&alpha;-DT NC||--||--
 +
|-
 +
|6||100bp ladder||--||--
 +
|}
 +
[[File:Igku_Sep4_Electrophoresis(N1).jpg]]<br>
 +
[[File:Igku_Sep4_Electrophoresis_N2_3.jpg]]<br>
 +
 +
</div>
 +
 +
===Gel Extraction===
 +
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
 +
1
 +
 +
{| class="wikitable"
 +
!Lane||DNA||Enzyme
 +
|-
 +
|1||100bp ladder||--
 +
|-
 +
|2||Pcon||SpeI&PstI
 +
|-
 +
|3||Pcon||SpeI&PstI
 +
|-
 +
|4||--||--
 +
|-
 +
|5||Plac||SpeI&PstI
 +
|-
 +
|6||Plac||SpeI&PstI
 +
|}
 +
 +
2
{| class="wikitable"
{| class="wikitable"
!Lane||DNA||Enzyme
!Lane||DNA||Enzyme
Line 57: Line 284:
|-
|-
|6||RBS-tetR-DT||XbaI&PstI
|6||RBS-tetR-DT||XbaI&PstI
 +
|}
 +
[[File:Igku Sep4 Electrophoresis(N2).jpg]]<br>
 +
[[File:Igku Sep4 Gel Extraction(N2) 2.jpg]]<br>
 +
{| class="wikitable"
 +
!Name||quantity||concentration[&micro;g/mL]||260/280||260/230
|-
|-
 +
|Pcon||--||13.2 ||1.99 ||-1.64
 +
|-
 +
|Plac||-- ||15.3 ||2.07 ||-0.39
 +
|-
 +
|Ptet||-- ||30.1||1.97 ||-0.97
 +
|-
 +
|RBS-tetR-DT||--||3.0||2.44 ||0.88
 +
|}
 +
</div>
-
 
+
===Gel Extraction===
-
 
+
<div class="experiment">
 +
<span class="author">No name</span></div>
 +
{| class="wikitable"
 +
!Lane||DNA||Enzyme
 +
|-
 +
|1||100bp ladder||--
 +
|-
 +
|2||spinach-DT||XbaI&PstI
 +
|-
 +
|3||spinach-DT ||XbaI&PstI
 +
|-
 +
|4||--||--
 +
|-
 +
|5||RBS-lacZ&alpha;-DT||XbaI&PstI
 +
|-
 +
|6||RBS-lacZ&alpha;-DT||XbaI&PstI
|}
|}
-
[[File:igku_xxbeforexx.xxx]]<br>
+
[[File:Igku Sep4 Gel Extraction(N3) 1.jpg]]<br>
-
[[File:igku_xxafterxx.xxx]]<br>
+
[[File:Igku Sep4 Gel Extraction(N3) 2.jpg]]<br>
{| class="wikitable"
{| class="wikitable"
!Name||quantity||concentration[&micro;g/mL]||260/280||260/230
!Name||quantity||concentration[&micro;g/mL]||260/280||260/230
|-
|-
-
|（´ω｀）|| || || ||
+
|spinach-DT||--||2.1||2.10||-0.05
|-
|-
-
|（ΦωΦ^）|| || || ||
+
|RBS-lacZ&alpha;-DT||--||3.3||2.59||-0.07
|}
|}
 +
</div>
===Master Plate===
===Master Plate===
Line 85: Line 342:
|-
|-
|}  
|}  
-
 
-
</div>
 
-
 
-
===Master Plate===
 
-
<div class="experiment">
 
-
<span class="author">Hirano</span>
 
{| class="wikitable"
{| class="wikitable"
!Number||Use LB plate
!Number||Use LB plate
Line 101: Line 352:
|-
|-
|4||JM109-4
|4||JM109-4
-
|-
 
|}  
|}  
-
 
-
 
-
 
</div>
</div>
Line 115: Line 362:
|-
|-
|8/9 BBa_J23100-3||Plusgrow medium(+Amp)
|8/9 BBa_J23100-3||Plusgrow medium(+Amp)
-
|-
 
-
|}
 
-
</div>
 
-
 
-
===Liquid Culture===
 
-
<div class="experiment">
 
-
<span class="author">Hirano</span>
 
-
{| class="wikitable"
 
-
!Sample||medium
 
|-
|-
|JM109-1(Master plate)||LB medium
|JM109-1(Master plate)||LB medium
-
|-
 
|}
|}
</div>
</div>

Latest revision as of 00:34, 27 September 2013

Contents

Sep 4

Ligation

No name

stateVectorInserterLigation High ver.2
experiment9/3 DT(EcoRI&XbaI)3.2µL9/3 RBS-lysis12.4µL2.7µL
experiment9/3 DT(EcoRI&XbaI) 3.0µL 9/3 RBS-lysis24.7µL 4.0µL
experiment9/3 DT(EcoRI&XbaI) 3.0µL 9/3 RBS-lysis38.8µL 4.0µL
experiment9/3 DT(EcoRI&XbaI) 3.0µL 9/3 aptamer12_1R(EcoRI&SpeI) 1.9µL 2.45µL
experiment9/2 pSB1C3(XbaI&PstI)10.6µL 9/3 pT181attenuator (XbaI& PstI)3.2µL 4.0µL
experiment9/2 pSB1C3(XbaI&PstI)9.6µL 9/3 pT181antisense (XbaI& PstI)2.1µL 4.0µL
experiment9/2 pSB1C3(EcoRI&SpeI)16.7µL 9/3 aptamer12_1R(EcoRI&SpeI) 2.0µL 4.0µL

incubate 16 °C 1 hour

Transformation

Tatsui

NameSampleCompetent CellsPlate
9/4 RBS-lysis1-DT 2µL20µLAmp
9/4 RBS-lysis2-DT 2µL20µLAmp
9/4 RBS-lysis3-DT 2µL20µLAmp
9/4 aptamer12_1R-DT2µL20µLAmp
9/4 pT181attenuator –pSB1C32µL20µLAmp
9/4 pT181antisense-pSB1C3 2µL20µLAmp
9/4 aptamer12_1R –pSB1C32µL20µLAmp
9/4 pSB4K5(2013plate5 56) 2µL20µLAmp
9/3 Mutagenesis product 1(KaiABC) 2µL20µLAmp
9/3 Mutagenesis product 2(NC) 2µL20µLAmp

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
DT193.11.931.81
Spinach-DT373.31.921.69
RBS-lysis1332.01.951.62

Master Plate

Hirano

NumberUse LB plate (+CP)
18/10 Plac(CP)-2
2 8/10 Plac(CP)-2
38/10 Plac(CP)-2
NumberUse LB plate (+Amp)
18/9 J23100(Amp)-1
28/9 J23100(Amp)-1
38/9 J23100(Amp)-1

Liquid Culture

Hirano

Samplemedium
8/10 Plac(CP)-2Plusgrow medium(+CP)
8/9 J23100(Amp)-1Plusgrow medium(+Amp)

PCR

Hirano

8/29 Pλ-RBS-luxI-DTKOD plus10x bufferdNTPMgSO4F primer (RBS-luxI-DT-cloning-fwd) R primer(RBS-luxI-DT-cloning-rev)MilliQtotal
0.3µL0.5µL 2.5µL 2.5µL 1.5µL 0.75µL 0.75µL 16.2µL 25µL
8/31 Pbad/araC-RBS-RFP(1)KOD plus10x bufferdNTPMgSO4F primer (Pbad/araC-pSB1C3-cloning-fond) R primer(Pbad/araC-cloning-rex)MilliQtotal
0.3µL0.5µL 2.5µL 2.5µL 1.5µL 0.75µL 0.75µL 16.2µL 25µL
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C65°C68°C--
5min10s30s3min10s30cycles

Restriction Enzyme Digestion

No name

8/21 PconstSpeIPstIBufferBSAMilliQtotal
2 cuts10.5µL1.0µL1.0µL3.0µL0.3µL14.2µL30 µL
NC0.5µL0µL0µ1.0µL0.1µL8.4µL10 µL
8/29 PlacSpeIPstIBufferBSAMilliQtotal
2 cuts13.0µL1.0µL1.0µL3.0µL0.3µL11.7µL30 µL
NC0.7µL0µL0µ1.0µL0.1µL 8.2µL10 µL
8/20 PtetSpeIPstIBufferBSAMilliQtotal
2 cuts9.0µL1.0µL1.0µL3.0µL0.3µL15.7µL30 µL
NC0.5µL0µL0µ1.0µL0.1µL 8.4µL10 µL
8/20 RBS-tetR-DTXbaIPstIBufferBSAMilliQtotal
2 cuts8.5µL1.0µL1.0µL3.0µL0.3µL16.2µL30 µL
NC0.5µL0µL0µ1.0µL0.1µL 8.4µL10 µL
spinach -DTXbaIPstIBufferBSAMilliQtotal
2 cuts5.4µL1.0µL1.0µL3.0µL3.0µL16.6µL30 µL
NC0.3µL0µL0µ1.0µL1.0µL 7.7µL10 µL
8/17 RBS-lacZα-DT XbaIPstIBufferBSAMilliQtotal
2 cuts11µL1.0µL1.0µL3.0µL3.0µL11µL30 µL
NC0.5µL0µL0µ1.0µL1.0µL7.5µL10 µL

Transformation

No name

NameSampleCompetent CellsTotalPlate
Ptrc KaiC1µL10µL11µLAmp
Ptrc KaiB1µL10µL11µLAmp
pSΩ 1µL10µL11µLAmp

Electrophoresis

No name
1

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2PconstSpeIPstI
3Pconst NC----
4PlacSpeIPstI
5Plac NC----
6100bp ladder----

2

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2PtetSpeIPstI
3Ptet NC----
4RBS-tetR-DTXbaIPstI
5RBS-tetR-DT NC----
6100bp ladder----

3

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2Spinach-DTXbaIPstI
3Spinach-DT NC----
4RBS-lacZα-DTXbaIPstI
5RBS-lacα-DT NC----
6100bp ladder----

Igku Sep4 Electrophoresis(N1).jpg
Igku Sep4 Electrophoresis N2 3.jpg

Gel Extraction

No name

1

LaneDNAEnzyme
1100bp ladder--
2PconSpeI&PstI
3PconSpeI&PstI
4----
5PlacSpeI&PstI
6PlacSpeI&PstI

2

LaneDNAEnzyme
1100bp ladder--
2PtetSpeI&PstI
3PtetSpeI&PstI
4----
5RBS-tetR-DTXbaI&PstI
6RBS-tetR-DTXbaI&PstI

Igku Sep4 Electrophoresis(N2).jpg
Igku Sep4 Gel Extraction(N2) 2.jpg

Namequantityconcentration[µg/mL]260/280260/230
Pcon--13.2 1.99 -1.64
Plac-- 15.3 2.07 -0.39
Ptet-- 30.11.97 -0.97
RBS-tetR-DT--3.02.44 0.88

Gel Extraction

No name
LaneDNAEnzyme
1100bp ladder--
2spinach-DTXbaI&PstI
3spinach-DT XbaI&PstI
4----
5RBS-lacZα-DTXbaI&PstI
6RBS-lacZα-DTXbaI&PstI

Igku Sep4 Gel Extraction(N3) 1.jpg
Igku Sep4 Gel Extraction(N3) 2.jpg

Namequantityconcentration[µg/mL]260/280260/230
spinach-DT--2.12.10-0.05
RBS-lacZα-DT--3.32.59-0.07

Master Plate

No name

NumberUse LB plate (+Amp)
48/9 BBa_J23100-3
58/9 BBa_J23100-4
68/9 BBa_J23100-5
NumberUse LB plate
1JM109-1
2JM109-2
3JM109-3
4JM109-4

Liquid Culture

Hirano

Samplemedium
8/9 BBa_J23100-3Plusgrow medium(+Amp)
JM109-1(Master plate)LB medium

Colony PCR

Hirano

Samplebase pair
8/9 BBa_J23100 3--
8/9 BBa_J23100 4--
8/9 BBa_J23100 5--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s30s30cycles
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