Template:Kyoto/Notebook/Sep 25

From 2013.igem.org

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(→RNA Extraction)
(→Ligation)
 
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Line 172: Line 172:
<span class="author">nakamoto>
<span class="author">nakamoto>
{| class="wikitable"
{| class="wikitable"
-
! ||9/24 Pcon-attenuator-aptamer-DT(&micro;L)||E(&micro;L)||S(&micro;L)||Buffer(&micro;L)||MilliQ(&micro;L)||total(&micro;L)
+
! ||9/24 Pcon-attenuator-aptamer-DT(&micro;L)||EcoRI(&micro;L)||SpeI(&micro;L)||Buffer(&micro;L)||MilliQ(&micro;L)||total(&micro;L)
|-
|-
|2 cuts||8.6||1||1||3||16.4||30
|2 cuts||8.6||1||1||3||16.4||30
Line 179: Line 179:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||9/11 DT(&micro;L)||E(&micro;L)||X(&micro;L)||Buffer(&micro;L)||BSA(&micro;L)||MilliQ(&micro;L)||total(&micro;L)
+
! ||9/11 DT(&micro;L)||EcoRI(&micro;L)||XbaI(&micro;L)||Buffer(&micro;L)||BSA(&micro;L)||MilliQ(&micro;L)||total(&micro;L)
|-
|-
|2 cuts||10.4||1||1||3||3||11.6||30
|2 cuts||10.4||1||1||3||3||11.6||30
Line 314: Line 314:
===Electrophoresis===
===Electrophoresis===
   
   
-
<!-- こっから -->
 
<div class="experiment">
<div class="experiment">
<span class="author">
<span class="author">
Line 322: Line 321:
|1||1kbp ladder||--||--
|1||1kbp ladder||--||--
|-
|-
-
|2||Pcon-pT181 attenuator-aptamer 12_1R-DT||E||S
+
|2||Pcon-pT181 attenuator-aptamer 12_1R-DT||EcoRI||SpeI
|-
|-
|3||Pcon-pT181 attenuator-aptamer 12_1R-DT||--||--
|3||Pcon-pT181 attenuator-aptamer 12_1R-DT||--||--
|-
|-
-
|4||DT||E||X
+
|4||DT||EcoRI||XbaI
|-
|-
|5||DT||--||--
|5||DT||--||--
Line 332: Line 331:
|}
|}
[[File:igku_92513.jpg]]<br>
[[File:igku_92513.jpg]]<br>
 +
===Gel Extraction===
===Gel Extraction===
-
<!-- こっから -->
+
 
<div class="experiment">
<div class="experiment">
-
<span class="author">nakamoto
+
<span class="author">Nakamoto
{| class="wikitable"
{| class="wikitable"
!Lane||DNA||Enzyme
!Lane||DNA||Enzyme
|-
|-
-
|1||DT||E+X
+
|1||DT||EcoRI+XbaI
|-
|-
-
|2||DT||E+X
+
|2||DT||EcoRI+XbaI
|-
|-
-
|3||Pcon-pT181 attenuator-aptamer12_1R-DT||E+S
+
|3||Pcon-pT181 attenuator-aptamer12_1R-DT||EcoRI+SpeI
|-
|-
-
|4||Pcon-pT181 attenuator-aptamer12_1R-DT||E+S
+
|4||Pcon-pT181 attenuator-aptamer12_1R-DT||EcoRI+SpeI
|-
|-
-
|5||Pcon-pT181 attenuator-aptamer12_1R-DT||E+S
+
|5||Pcon-pT181 attenuator-aptamer12_1R-DT||EcoRI+SpeI
|}
|}
[[File:igku_92514.jpg]]<br>
[[File:igku_92514.jpg]]<br>
Line 356: Line 356:
|Pcon-pT181 attenuator-aptamer12_1R-DT(EcoRI&SpeI)||11.8||1.80||0.46
|Pcon-pT181 attenuator-aptamer12_1R-DT(EcoRI&SpeI)||11.8||1.80||0.46
|-
|-
-
|DT(E+X)||42.6||1.84||0.98
+
|DT(EcoRI+XbaI)||42.6||1.84||0.98
|}
|}
Line 377: Line 377:
!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;L||name||xx &micro;L||xx &micro;L
+
|experiment||9/24 pSB1C3(EcoRI+Spel)||10&micro;L||9/25 PconpT181attenuator-aptamer12-1R-DT(EcoRI+Spel)||5.9&micro;L||8&micro;L
-
|-
+
-
|NC||name||xx &micro;L||name||xx &micro;L||xx &micro;L
+
|}
|}
</div>
</div>
Line 385: Line 383:
===PCR===
===PCR===
-
<!-- ここから -->
 
<div class="experiment">
<div class="experiment">
-
<span class="author">hirano
+
<span class="author">Hirano
{| class="wikitable"
{| class="wikitable"
!800pg/μL DT||KOD plus||10x buffer||dNTP||MgSO4||1C3_GG2_fwd||iC3_GG1_rev||MilliQ||total
!800pg/μL DT||KOD plus||10x buffer||dNTP||MgSO4||1C3_GG2_fwd||iC3_GG1_rev||MilliQ||total
Line 402: Line 399:
===Transformation===
===Transformation===
-
<!-- こっから -->
 
<div class="experiment">
<div class="experiment">
<span class="author">Tatsui</span>
<span class="author">Tatsui</span>
{| class="wikitable"
{| class="wikitable"
-
!Name||Sample||Competent Cells||Total||Plate
+
!Name||Plate
|-
|-
-
|aptamer12-1R-DT||&micro;L||&micro;L||&micro;L||CP
+
|aptamer12-1R-DT||CP
|-
|-
-
|pSB1C3(EcoRI+SpeI)+pT181attenuator(EcoRI+Spal)||&micro;L||&micro;L||&micro;L||CP
+
|pSB1C3(EcoRI+SpeI)+pT181attenuator(EcoRI+Spal)||CP
|-
|-
-
|Pcon-pT181attenuator(EcoRI+XbaI)+RBS-GFP-DT(EcoRI+SpeI)||&micro;L||&micro;L||&micro;L||Amp
+
|Pcon-pT181attenuator(EcoRI+XbaI)+RBS-GFP-DT(EcoRI+SpeI)||Amp
|-
|-
-
|Pcon-RBS-GFP-DT(EcoRI+XbaI)+Pcon-RBS-tetR-DT(EcoRI+SpeI)||&micro;L||&micro;L||&micro;L||Amp
+
|Pcon-RBS-GFP-DT(EcoRI+XbaI)+Pcon-RBS-tetR-DT(EcoRI+SpeI)||Amp
|-
|-
-
|pSB1C3(EcoRI+SpeI)+Pcon-pT181attenuator-aptamer12-1R-DT(EcoRI+SpeI)||&micro;L||&micro;L||&micro;L||CP
+
|pSB1C3(EcoRI+SpeI)+Pcon-pT181attenuator-aptamer12-1R-DT(EcoRI+SpeI)||CP
|}
|}
</div>
</div>
-
<!-- ここまでをコピーしてね -->
 
===Electrophoresis===
===Electrophoresis===
Line 449: Line 444:
Concentrate PCR product by evaporator until volume become less than 3&micro;L, add all sulution and pipetting. </div>
Concentrate PCR product by evaporator until volume become less than 3&micro;L, add all sulution and pipetting. </div>
{| class="wikitable"
{| class="wikitable"
-
! ||9/25 DT(2-1)||10*cut smart Buffer||BsaI-HF||MilliQ||total
+
!9/25 DT(2-1)||10*cut smart Buffer||BsaI-HF||MilliQ||total
|-
|-
-
| ||3.0 &micro;L||2.0 &micro;L||0.3 &micro;L||14.7 &micro;L||20.0 &micro;L||
+
|3.0 &micro;L||2.0 &micro;L||0.3 &micro;L||14.7 &micro;L||20.0 &micro;L
|}
|}
Incubate 37&deg;C 12hour
Incubate 37&deg;C 12hour
-
</div>
 
===Liquid Culture===
===Liquid Culture===
-
<!-- ここから -->
 
<div class="experiment">
<div class="experiment">
-
<span class="author">hirano</span>
+
<span class="author">Hirano</span>
{| class="wikitable"
{| class="wikitable"
!Sample||medium
!Sample||medium
Line 570: Line 563:
|RNase-frec water||4 &micro;L
|RNase-frec water||4 &micro;L
|}
|}
-
</div>
+
Incubate 42&deg;C 2min, then on ice immediately<BR>
-
Incubate 42&deg;C 2min, then on ice immediately</div>
+
Add <BR>
-
Add </div>
+
{| class="wikitable"
{| class="wikitable"
!5*Quantiscript Reverse Transcriptase||0.5 &micro;L
!5*Quantiscript Reverse Transcriptase||0.5 &micro;L
Line 580: Line 572:
|RT Primer Mix||0.5&micro;L
|RT Primer Mix||0.5&micro;L
|}
|}
-
</div>
 
Incubate 42&deg;C 15min, then incubate 95&deg;C 3min
Incubate 42&deg;C 15min, then incubate 95&deg;C 3min
 +
</div>
===Electrophoresis===
===Electrophoresis===
Line 600: Line 592:
===Ligation===
===Ligation===
-
+
 
-
<!-- こっから -->
+
<div class="experiment">
<div class="experiment">
-
<span class="author">tatsui</span>
+
<span class="author">Tatsui</span>
{| class="wikitable"
{| class="wikitable"
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
|-
|-
-
|experiment||9/13 Pcon-pT181-attenuator(SpeI+PstI)||3.5&micro;L||9/25 RBS-GFP-DT(X+P)||13.5&micro;L||8.5 &micro;L
+
|experiment||9/13 Pcon-pT181-attenuator(SpeI&PstI)||3.5&micro;L||9/25 RBS-GFP-DT(XbaI&PstI)||13.5&micro;L||8.5 &micro;L
|}
|}
</div>
</div>
-
<!-- ここまでをコピーしてね -->
 

Latest revision as of 19:26, 27 September 2013

Contents

Sep 25

Electrophoresis

nakamoto

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2Pcon-antisense (1C3-66E-fwd A2-662 rev)----
3DT (1C3-661-fwd 1C3 662 rev)----
4spinach-DT(1C3-661-fwd 1C3 662 rev)----
5Pcon-antisense-spinach-DT(1C3-66E fwd A2-661 rev)----
6100bp ladder----

Igku 9251a.jpg

LaneSampleEnzyme1Enzyme2
2100bp ladder----
3Ptet (1C3 66E fwd-1C3 660 rev)----
4Pcon-attenuator(1C3-660 fwd A2-661 rev)----
5DT(1C3 662 fwd-1C3 661 rev)----
6Pcon-attenuator-aptamer-DT(1C3 66E fwd-A2-661-rev)----
7100bp ladder----

Igku 9251b.jpg

Colony PCR

Nakamoto

Samplebase pair
9/23 Pcon-PT181 attenuator-aptamer12-1R-DT-(5~12)859
9/23 Pcon-PT181 attenuator-RBS-GFP-DT-(5~8)1527
9/23 Pcon-PT181 attenuator-pSB1C3-(5~8)601
PreDenatureDenatureAnnealingExtensioncycle
94 °C94 °C55 °C68 °C--
2 min30 s30 s1min5s30 cycles


Electrophoresis

no name

LaneSampleEnzyme1Enzyme2
1Pcon-PT181 attenuator-aptamer12-1R-DT-5----
2Pcon-PT181 attenuator-aptamer12-1R-DT-6----
3Pcon-PT181 attenuator-aptamer12-1R-DT-7----
4Pcon-PT181 attenuator-aptamer12-1R-DT-8----
5Pcon-PT181 attenuator-aptamer12-1R-DT-9----
6Pcon-PT181 attenuator-aptamer12-1R-DT-10----
7Pcon-PT181 attenuator-aptamer12-1R-DT-11----
8Pcon-PT181 attenuator-aptamer12-1R-DT-12----
91kbp ladder----
10Pcon-PT181 attenuator-RBS-GFP-DT-5----
11Pcon-PT181 attenuator-RBS-GFP-DT-6----
12Pcon-PT181 attenuator-RBS-GFP-DT-7----
13Pcon-PT181 attenuator-RBS-GFP-DT-8----
14Pcon-PT181 attenuator-pSB1C3-5----
15Pcon-PT181 attenuator-pSB1C3-6----
16Pcon-PT181 attenuator-pSB1C3-7----
17Pcon-PT181 attenuator-pSB1C3-8----

Igku 9252.jpg

Transformation

kojima and nakamoto

NameSampleCompetent CellsTotalPlate
Plac+tet aptamer12+Pcon tetR-DT+Ptet+RBS-GFP-DT1µL10µL11µL--
Pcon-pT181attenuator-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT1µL10µL11µL--
Pcon-tet aptamer12-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT1µL10µL11µL--
Pcon-Spinach-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT1µL10µL11µL--
Pcon-pT181antisense+DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT1µL10µL11µL--
Pcon-pT181attenuator+tet aptamer12-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT1µL10µL11µL--
Pcon tetR-DT+Ptet+RBS-GFP-DT1µL10µL11µL--
Pcon tetR-DT+Pcon-GFP-DT+RBS-GFP-DT1µL10µL11µL--
Pcon-tet aptamer12-DT+Pcon-pT181attenuator+RBS-GFP-DT1µL10µL11µL--
Pcon-pT181attenuator-DT+Pcon-pT181attenuator+RBS-GFP-DT1µL10µL11µL--
Pcon-tet aptamer12-DT+Ptet-pT181antisense+Spinach-DT+Pcon-RBS-GFP-DT+RBS-GFP-DT1µL10µL11µL--
Pcon-tet aptamer12-DT+Pcon-pT181antisense+Spinach-DT+Pcon-pT181attenuator+RBS-GFP-DT1µL10µL11µL--
Pcon-tet aptamer12-DT+Ptet-pT181antisense+Spinach-DT+Pcon-pT181attenuator+RBS-GFP-DT1µL10µL11µL--

NameSampleCompetent CellsTotalPlate
aptamer12-DT(EcoRI+XbaI)+Pcon-pT181attenuator(EcoRI+SpeI)2µL20µL22µL--
Ptet(SpeI+PstI)+RBS-GFP-DT(XbaI+PstI))2µL20µL22µL--

Ligation

nakamoto

stateVectorInserterLigation High ver.2
experiment9/25 aptamer12_1R-DT(EcoRI&XbaI)4.5 µLPcon-pT181 attenuator(EcoRI&SpeI)7.7 µL6.1 µL
experiment9/17 pSB1C3(EcoRI&SpeI)2.0 µL9/3 pT181 attenuator(EcoRI&SpeI)14 µL8 µL
experiment9/25 Pcon-pT181 attenuator(EcoRI&XbaI)4.5 µL9/25 RBS-GFP-DT(EcoRI&SpeI)19.3 µL11.9 µL
experiment9/25 Pcon-RBS-GFP-DT(EcoRI&XbaI)4.2 µLPcon-RBS-tetR-DT(EcoRI&SpeI)20 µL12.1 µL

Restriction Enzyme Digestion

nakamoto>

9/24 Pcon-attenuator-aptamer-DT(µL)EcoRI(µL)SpeI(µL)Buffer(µL)MilliQ(µL)total(µL)
2 cuts8.611316.430
NC0.40018.610
9/11 DT(µL)EcoRI(µL)XbaI(µL)Buffer(µL)BSA(µL)MilliQ(µL)total(µL)
2 cuts10.4113311.630
NC0.500117.510

Liquid Culture

Kojima

Samplemedium
DT --
pT181attenuator --
Pcon-RBS-tetR-DT --

Gel Extraction

Kojima

LaneDNAEnzyme
1100bp ladder--
2Pcon-pT181antisense--
3
4
5----
6DT--
7
8
9----
10Spinach-DT--
11
12

Igku 9253.jpg
Igku 9254.jpg

LaneDNAEnzyme
1100bp ladder--
3Pcon-antisense-spinach-DT(E-1A)--
4Pcon-antisense-spinach-DT(E-1A)--
5Pcon-antisense-spinach-DT(E-1A)--

Igku 9255.jpg
Igku 9256.jpg

LaneDNAEnzyme
7100bp ladder--
9Ptet(E-0)--
10Ptet(E-0)--
11Ptet(E-0)--

Igku 9257.jpg
Igku 9258.jpg

LaneDNAEnzyme
13100bp ladder--
15Pcon-attenuator(0-1A)--
16Pcon-attenuator(0-1A)--
17Pcon-attenuator(0-1A)--

Igku 9259.jpg
||-- Igku 92510.jpg

LaneDNAEnzyme
19100bp ladder--
21Pcon-attenuator-aptamer-DT(E-1A)--
22Pcon-attenuator-aptamer-DT(E-1A)--
23Pcon-attenuator-aptamer-DT(E-1A)--

Igku 92511.jpg
Igku 92512.jpg

Nameconcentration[µg/mL]260/280260/230
DT(2-1)24.91.730.37
Ptet(E-0)12.81.590.90
Pcon-antisense(E-2A)16.51.721.07
Pcon-attenuator-aptamer-DT(E-1A)22.11.750.94
Pcon-antisense-spinach-DT(E-1A)31.01.781.15
spinach-DT(1-2)23.41.851.11
Pcon-attenuator(0-1A)22.51.760.46
DT(1-2)25.11.781.21

Electrophoresis

LaneSampleEnzyme1Enzyme2
11kbp ladder----
2Pcon-pT181 attenuator-aptamer 12_1R-DTEcoRISpeI
3Pcon-pT181 attenuator-aptamer 12_1R-DT----
4DTEcoRIXbaI
5DT----

Igku 92513.jpg

Gel Extraction

Nakamoto

LaneDNAEnzyme
1DTEcoRI+XbaI
2DTEcoRI+XbaI
3Pcon-pT181 attenuator-aptamer12_1R-DTEcoRI+SpeI
4Pcon-pT181 attenuator-aptamer12_1R-DTEcoRI+SpeI
5Pcon-pT181 attenuator-aptamer12_1R-DTEcoRI+SpeI

Igku 92514.jpg
Igku 92515.jpg

Nameconcentration[µg/mL]260/280260/230
Pcon-pT181 attenuator-aptamer12_1R-DT(EcoRI&SpeI)11.81.800.46
DT(EcoRI+XbaI)42.61.840.98

Liquid Culture

No name

Samplemedium
Pcon-pT181anntisense-spinach-DT(4k5)-

Ligation

Nakamoto and Tatsui

stateVectorInserterLigation High ver.2
experiment9/24 pSB1C3(EcoRI+Spel)10µL9/25 PconpT181attenuator-aptamer12-1R-DT(EcoRI+Spel)5.9µL8µL

PCR

Hirano

800pg/μL DTKOD plus10x bufferdNTPMgSO41C3_GG2_fwdiC3_GG1_revMilliQtotal
10.52.52.51.50.750.7515.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s24s30cycles

Transformation

Tatsui

NamePlate
aptamer12-1R-DTCP
pSB1C3(EcoRI+SpeI)+pT181attenuator(EcoRI+Spal)CP
Pcon-pT181attenuator(EcoRI+XbaI)+RBS-GFP-DT(EcoRI+SpeI)Amp
Pcon-RBS-GFP-DT(EcoRI+XbaI)+Pcon-RBS-tetR-DT(EcoRI+SpeI)Amp
pSB1C3(EcoRI+SpeI)+Pcon-pT181attenuator-aptamer12-1R-DT(EcoRI+SpeI)CP

Electrophoresis

Hirano

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2DT(2-1)----

Igku 92516.jpg

Column Refining

Nameconcentration[µg/mL]260/280260/230
8/25 DT(2-1)------

Restriction Enzyme Digestion

Concentrate PCR product by evaporator until volume become less than 3µL, add all sulution and pipetting.
9/25 DT(2-1)10*cut smart BufferBsaI-HFMilliQtotal
3.0 µL2.0 µL0.3 µL14.7 µL20.0 µL

Incubate 37°C 12hour

Liquid Culture

Hirano

Samplemedium
Pcon-GFP-DTLB(Amp)
RBS-GFP-DTLB(CP)
Pcon-spinach-DTLB(Amp)
Pcon-tetRaptamer-DTLB(Amp)
Pcon-attenuator-DTLB(Amp)
spinach-DTLB(CP)
Pcon-antisense-spinach-DTLB(Amp)

Plating

Hirano

Mix Liquid Culture 1mL and LB(+Amp), incubate 37°C 30min
OD600-1.445
SampleUse plateThe number of plate
Pcon-GFP-DTM9(+Amp)3
Pcon-GFP-DTLB(+Amp)3

</div> incubate 37°C

Colony PCR

No name

Samplebase pair
9/24 GGA-1(1~8)3177
9/24 GGA-2(1~8)3189
9/24 GGA-3(1~8)2989
9/24 GGA-4(1~8)2939
9/24 GGA-7(1)2489
GGA-16(1)2717
RBS-GFP-DT--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s3min15s30cycles

RNA Extraction

Nakamoto
sumple 0.25mL
Add 1mLISOGEN-LS
Lysis or homogenization
Store for 5 min, at room temperature
Add 0.2mL Chloroform
Shake vigorously for 15sec.
Store for 2~3min.at room temperature
Centrifuge 12K*g for 15min. at 4°C
Extract aqueous phase
Add 0.5mL isopropanol
Store for 5~10min. at room temperature
Centrifuge 12K*g for 10min. at 4°C
Remove aqueous phase
Add at least 1mL 70% ethanol
Vortex
Centrifuge 7.5K*g for 5min. at 4°C
Remove aqueous phase
Dry briefly
Add ddH2O, TE(pH8.0) or 0.5% SDS
Dissolve
Total RNA solution

DNAconcentration[µg/mL]260/280260/230
Pcon-RBS-GFP-DT27831.951.48

Add 91.2µL, Store at freezer
Total 250µL</div>

cDNA　Synthesis

Nakamoto

volume7 µL
7*cDNA Wipcout Buffer1 µL
Template RNA(9/25 Pcon-RBS-GFP-DT)3 µL
RNase-frec water4 µL

Incubate 42°C 2min, then on ice immediately
Add

5*Quantiscript Reverse Transcriptase0.5 µL
5*Quantiscript RT Buffer2 µL
RT Primer Mix0.5µL

Incubate 42°C 15min, then incubate 95°C 3min

Electrophoresis

Nakamoto

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2Pcon-RBS-GFP-DT(RNA)----

Igku 92517.jpg

Ligation

Tatsui

stateVectorInserterLigation High ver.2
experiment9/13 Pcon-pT181-attenuator(SpeI&PstI)3.5µL9/25 RBS-GFP-DT(XbaI&PstI)13.5µL8.5 µL

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