Template:Kyoto/Notebook/Sep 27

From 2013.igem.org

(Difference between revisions)
(→Gel Extraction)
(→Sep 27)
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|attenuator(1C3)||--||2||--||20||22
|attenuator(1C3)||--||2||--||20||22
|}
|}
 +
 +
===Miniprep===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{|class="wikitable"
 +
!DNA||concentration[&micro;g/mL]||260/280||260/230
 +
|-
 +
|Pcon-RBS－GFP-DT||137.6||1.95||1.78
 +
|-
 +
|RBS-GFP-DT||77.0||2.06||1.96
 +
|-
 +
|Pcon-PT181 attenuator|231.0||1.89||1.89
 +
|-
 +
|Pcon-antisense-Spinach-DT(2)||130.0||1.56||1.62
 +
|-
 +
|Pcon-aptamer-DT||218.8||1.92||1.72
 +
|-
 +
|Pcon-attenuator-DT||180.7||1.95||1.78
 +
|-
 +
|Pcon-Spinach-DT||177.6||1.96||1.06
 +
|-
 +
|Pcon-antisense-Spinach-DT(1)||181.1||1.96||1.92
 +
|-
 +
|Pcon-antisense||152.9||2.02||1.96
 +
|-
 +
|Pcon-RBS+ezR-DT||164.1||1.93||1.91
 +
|}
 +
</div>
 +
 +
===Restriction Enzyme Digestion===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
! ||9/27 RBS-GFP-DT||XbaI||PstI||Buffer||BSA||MilliQ||total
 +
|-
 +
|2 cuts||12&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||20&micro;L||40&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
 +
|}
 +
{| class="wikitable"
 +
! ||9/27 Pcon-tetR-DT||EcoRI||PstI||Buffer||MilliQ||total
 +
|-
 +
|2 cuts||12.2&micro;L||1&micro;L||1&micro;L||3&micro;L||12.8&micro;L|| 30&micro;L
 +
|-
 +
|NC||0.6&micro;L||0&micro;L||0&micro;L||1&micro;L||8.4&micro;L||10&micro;L
 +
|}
 +
</div>
 +
 +
===RNA Extraction===
 +
<div class="experiment">
 +
<span class="author">Nakamoto</span>
 +
{| class="wikitable"
 +
!Name||concentration[&micro;g/mL]||260/280||260/230
 +
|-
 +
|9/27 Pcon-RBS-GFP-DT||686.6||1.99||1.34
 +
|-
 +
|9/27 Pcon-RBS-GFP-DT (+High salt)||127.6||1.76||0.27
 +
|}
 +
</div>
 +
 +
===DNA sythesis===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!genome DNA||DNA volume||genome DNA wipeout buffer||RNase freewater||RT||RT buffer||Primer Mix||total
 +
|-
 +
|9/27 Pcon-RBS-GFP-DT||0.8&micro;L||1&micro;L||5.2&micro;L ||0.5&micro;L ||2&micro;L||0.5&micro;L ||10&micro;L
 +
|-
 +
|9/27 pcon-RBS-GFP-DT(+high salt)||2.5&micro;L||1&micro;L||3.5&micro;L ||0.5&micro;L ||2&micro;L ||0.5&micro;L ||10&micro;L
 +
|-
 +
|9/27 Pcon-RBS-GFP-DT(revised concentration)|| 0.5&micro;L||1&micro;L||5.5&micro;L||0.5&micro;L||2&micro;L||0.5&micro;L||10&micro;L
 +
|}
 +
</div>
 +
===Electrophoresis===
 +
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Lane||Sample
 +
|-
 +
|1||1kb ladder
 +
|-
 +
|2||Pcon-RBS-GFP-DT
 +
|-
 +
|3|| Pcon-RBS-GFP-DT
 +
|}
 +
[[File:igku_0927_E2]]<br>
 +
</div>
 +
===Liquid Culture===
===Liquid Culture===
-
<!-- ここから -->
 
<div class="experiment">
<div class="experiment">
<span class="author">No name</span>
<span class="author">No name</span>

Revision as of 13:27, 27 September 2013

Contents

Sep 27

Gel Extraction

Kojima

LaneDNAEnzyme
11kbp ladder--
2pSB1C3EcoRI+SpeI
3
4
6Pcon-attenuator-aptmer-DTEcoRI+SpeI
7
8
10Pcon-attenuator-aptmer-DTEcoRI+SpeI
11
12

Igku 9272.jpg
Igku 9273.jpg

Nameconcentration[µg/mL]260/280260/230
Pcon-attenuator-DT(EcoRI+SpeI)6.81.980.43
pT181-attenuator(EcoRI+SpeI)7.51.840.06
pSB1C3(EcoRI+SpeI)17.71.970.71

Gel Extraction

No name

LaneDNAEnzyme
11kbp ladder--
3pSB4K5EcoRI+PstI
4pSB4K5EcoRI+PstI
5pSB4K5EcoRI+PstI
7pSB6A1EcoRI+PstI
8pSB6A1EcoRI+PstI
9pSB6A1EcoRI+PstI

Igku 9274.jpg
Igku 9275.jpg

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1pSB4K5EcoRIPstI
2pSB4K5--
31kbp ladder--
4Pcon-attenuatorEcoRISpeI
5Pcon-attenuator--


LaneSampleEnzyme1Enzyme2
1RBS-GFP-DTXbaIPstI
2RBS-GFP-DT--
3RBS-GFP-DTEcoRIXbaI
41kbp ladder--
5PtetEcoRISpeI
6Ptet--

Igku 0927 G5.jpg

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder--
3PtetEcoRI+SpeI
4PtetEcoRI+SpeI
5PtetEcoRI+SpeI

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

LaneDNAEnzyme
11kbp ladder--
3Pcon-antisense-aptamer-DTXbaI+PstI
4Pcon-antisense-aptamer-DTXbaI+PstI
5Pcon-antisense-aptamer-DTXbaI+PstI

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

LaneDNAEnzyme
11kbp ladder--
3RBS-GFP-DTEcoRI+XbaI
4RBS-GFP-DTEcoRI+XbaI
5RBS-GFP-DTEcoRI+XbaI

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

LaneDNAEnzyme
11kb ladder--
2Pcon-attenuatorEcoRI+SpeI
3Pcon-attenuatorEcoRI+SpeI
4Pcon-attenuatorEcoRI+SpeI
6pSB4K5EcoRI+PstI
7pSB4K5EcoRI+PstI
8pSB4K5EcoRI+PstI
10RBS-GFP-DTXbaI+PstI
11RBS-GFP-DTXbaI+PstI
12RBS-GFP-DTXbaI+PstI

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

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

Liquid Culture

No name

Samplemedium
Pcon-GFPAmp
RBS-GFP-DTCP
Pcon-attenuatorAmp
Pcon-attenuator-aptamer-DTAmp
Pcon-tetR-DTAmp

Ligation

No name

stateVectorInserterLigation High ver.2
experiment9/17 pSB1C3 (EcoRI+SpeI) 10.0ng/µL10µL9/26 pT181 attenuator(EcoRI+SpeI) 7.5ng/µL1.3µL3.5 µL
experiment9/25 DT (EcoRI+XbaI) 42.8ng/µL2.3µL9/22 Pcon-pT181 antisense(EcoRI+SpeI) 46.7ng/µL1.5µL1.9 µL
experiment9/27 pSB4K5(EcoRI+PstI) 12.9ng/µL7.8µL9/10 Pcon-attenuator(EcoRI+SpeI) 13.8ng/µL8.0µL3.5 µL
experiment9/27 pSB4K5(EcoRI+PstI) 12.9ng/µL7.8µL9/25 RBS-GFP-DT(XbaI+PstI) 13.9ng/µL12µL3.5 µL
experiment9/8 pSB4K5 (EcoRI+SpeI) 17.6ng/µLxx µL8/21 Pcon-GFP-DT(EcoRI+SpeI) 11ng/µL12.6µLxx µL

Liquid Culture

Hirano

Samplemedium
Pcon-spinach-DTplusgrow Amp
Pcon-tetRaptamer-DTplusgrow Amp　
Pcon-antisense-spinach-DTplusgrow Amp
spinach-DTplusgrow CP

RNA Extraction

Nakamoto

Nameconcentration[µg/mL]260/280260/230
9/26 Pcon-GFP-DT423.01.831.46
9/26 Pcon-GFP-DT K 298.71.821.50

Electrophoresis

No name

LaneSample
19/27 Pcon-GFP-DT(RNA)
21kb ladder
39/27 Pcon-GFP-DT K(RNA)

Igku 9271.jpg

Miniprep

Tatsui

DNA
Pcon-aptamer-DT
Pcon-spinach-DT
Pcon-antisense
Pcon-tetR-DT
Pcon-attenuator-DT
Pcon-antisense-spinach-DT

Transformation

Hirano

Name1Name2Sample1(µL)Sample2(µL)Competent Cells(µL)Total(µL)
Pcon-attenuator+RBS-GFP-DT(4K5)Pcon-attenuator-DT(1A2)213033
Pcon-attenuator+RBS-GFP-DT(4K5)Pcon-antisense-spinach-DT(1A2)213033
Pcon-attenuator+RBS-GFP-DT(4K5)Pcon-tetR aptamer-DT(1A2)213033
Pcon-RBS-GFP-DT(4K5)Pcon-attenuator-DT(1A2)213033
Pcon-RBS-GFP-DT(4K5)Pcon-antisense-spinach-DT(1A2)213033
Pcon-RBS-GFP-DT(4K5)Pcon-tetR aptamer-DT(1A2)213033
Pcon-attenuator+RBS-GFP-DT(4K5)--2--2022
Pcon-RBS-GFP-DT(4K5)--2--2022
attenuator(1C3)--2--2022

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
Pcon-RBS－GFP-DT137.61.951.78
RBS-GFP-DT77.02.061.96
231.01.891.89
Pcon-antisense-Spinach-DT(2)130.01.561.62
Pcon-aptamer-DT218.81.921.72
Pcon-attenuator-DT180.71.951.78
Pcon-Spinach-DT177.61.961.06
Pcon-antisense-Spinach-DT(1)181.11.961.92
Pcon-antisense152.92.021.96
Pcon-RBS+ezR-DT164.11.931.91

Restriction Enzyme Digestion

No name

9/27 RBS-GFP-DTXbaIPstIBufferBSAMilliQtotal
2 cuts12µL1µL1µL3µL3µL20µL40µL
NC1.3µL0µL0µL1µL1µL6.7µL10µL
9/27 Pcon-tetR-DTEcoRIPstIBufferMilliQtotal
2 cuts12.2µL1µL1µL3µL12.8µL 30µL
NC0.6µL0µL0µL1µL8.4µL10µL

RNA Extraction

Nakamoto

Nameconcentration[µg/mL]260/280260/230
9/27 Pcon-RBS-GFP-DT686.61.991.34
9/27 Pcon-RBS-GFP-DT (+High salt)127.61.760.27

DNA sythesis

No name

genome DNADNA volumegenome DNA wipeout bufferRNase freewaterRTRT bufferPrimer Mixtotal
9/27 Pcon-RBS-GFP-DT0.8µL1µL5.2µL 0.5µL 2µL0.5µL 10µL
9/27 pcon-RBS-GFP-DT(+high salt)2.5µL1µL3.5µL 0.5µL 2µL 0.5µL 10µL
9/27 Pcon-RBS-GFP-DT(revised concentration) 0.5µL1µL5.5µL0.5µL2µL0.5µL10µL

Electrophoresis

No name

LaneSample
11kb ladder
2Pcon-RBS-GFP-DT
3 Pcon-RBS-GFP-DT

File:Igku 0927 E2


Liquid Culture

No name

Samplemedium
Pcon spinach DT plusgrow 2µL(Amp)
Pcon aptamer DT plusgrow 2µL(Amp)
Pcon antisense Spinach DT plusgrow 2µL(Amp)
Spinach DTplusgrow 4µL(CP)　

RT PCR

No name

genome DNAKOD plus10x buffer for KOD-plus-ver.22nM dNTPs25mM MgSO4F primerR primerMilliQtotal
0.512.52.51.50.750.7515.525
PreDenatureDenatureAnnealingExtensioncycle
　94°C　98°C　49.8°C　68°C--
2min　10sec　30sec　10sec　

Electrophoresis

No name

LaneSample
1Pcon-GFP-DT(GFP)
2Pcon-GFP-DT(internal)
3Pcon-GFP-DT(HS)(GFP)
4Pcon-GFP-DT(internal)

File:Igku 927x.jpg

LaneSample
1Pcon-GFP-DT(GFP)
2Pcon-GFP-DT(internal)
3100bp ladder

File:Igku 927y.jpg

Digesting cut check

No name

LaneSample
1RBS-GFP-DT(XbaI&PstI)
2RBS-GFP-DT NC(XbaI&PstI)
31kbp ladder
4Pcon-RBS-tetR-DT(EcoRI&SpeI)
5Pcon-RBS-tetR-DT NC(EcoRI&SpeI)

Igku 927 E3.jpg

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