Template:Kyoto/Notebook/Sep 16

From 2013.igem.org

(Difference between revisions)
(Sep 16)
(PCR)
 
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!Lane||DNA||Enzyme
!Lane||DNA||Enzyme
|-
|-
-
|1||1kb ladder
+
|1||1kb ladder||
|-
|-
|2||pT181 attenuator||XbaI+PstI
|2||pT181 attenuator||XbaI+PstI
Line 57: Line 57:
|-
|-
|}
|}
-
[[File:igku_xxbeforexx.xxx]]<br>
+
[[File:igku_9161.png]]<br>
-
[[File:igku_xxafterxx.xxx]]<br>
+
[[File:igku_9161a.png]]<br>
{| class="wikitable"
{| class="wikitable"
!Name||concentration[&micro;g/mL]||260/280||260/230
!Name||concentration[&micro;g/mL]||260/280||260/230
Line 105: Line 105:
{| class="wikitable"
{| class="wikitable"
{| class="wikitable"
{| class="wikitable"
-
!DNA||9/6pT181 anti psB1C3(1)||KOD plus||10x buffer||dNTP||MgSO4||GG1_1C3_fwd primer||GG2_1C3_rev primer||MilliQ||total
+
!DNA||9/6pT181 antisense psB1C3(1)||KOD plus||10x buffer||dNTP||MgSO4||GG1_1C3_fwd primer||GG2_1C3_rev primer||MilliQ||total
|-
|-
|400pg||1||0.5||2.5||2.5||1.5||0.75||0.75||15.5||25
|400pg||1||0.5||2.5||2.5||1.5||0.75||0.75||15.5||25
Line 164: Line 164:
|8||NC
|8||NC
|}
|}
-
[[File:igku_xxxxxx.xxx]]<br>
+
[[File:igku_9162.png]]<br>
===Restriction Enzyme Digestion===
===Restriction Enzyme Digestion===
Line 211: Line 211:
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
|-
|-
-
|experiment||Pcon-pT181anti-spinach-DT(XbaI+PstI)||5.0&micro;g/mL||pSB4K5(XbaI+PstI)||18.1&micro;g/mL||xx &micro;g/mL
+
|experiment||Pcon-pT181antisense-spinach-DT(XbaI+PstI)||5.0&micro;g/mL||pSB4K5(XbaI+PstI)||18.1&micro;g/mL||xx &micro;g/mL
|-
|-
|experiment||pC181 attenuator(XbaI+PstI)||8.2&micro;g/mL||pSB1C3(XbaI+PstI)||26.0&micro;g/mL||xx &micro;g/mL
|experiment||pC181 attenuator(XbaI+PstI)||8.2&micro;g/mL||pSB1C3(XbaI+PstI)||26.0&micro;g/mL||xx &micro;g/mL
 +
|}
 +
 +
===Transformation===
 +
<!-- こっから -->
 +
<div class="experiment">
 +
<span class="author">tatsui</span>
 +
{| class="wikitable"
 +
!Name||Sample||Competent Cells||Total||Plate
 +
|-
 +
|Pcon+RBS-laczα-DT|| &micro;L|| &micro;L|| &micro;L||
 +
|-
 +
|Pcon-pT181 attenuator-DT+pSB1C3|| &micro;L|| &micro;L|| &micro;L||
 +
|-
 +
|pT181 attenuator(EcoRI&SpeI)+pSB1C3|| &micro;L|| &micro;L|| &micro;L||
 +
|-
 +
|Pcon-aptamer12_1R-DT(EcoRI&SpeI)+pSB1C3|| &micro;L|| &micro;L|| &micro;L||
 +
|-
 +
|Pcon-spinach-DT(EcoRI&SpeI)+pSB1C3|| &micro;L|| &micro;L|| &micro;L||
 +
|-
 +
|Pcon-RBS-tetR-DT(EcoRI&SpeI)+pSB1C3|| &micro;L|| &micro;L|| &micro;L||
 +
|-
 +
|Pcon-pT181 antisense-spinach-DT(XbaI&PstI)+pSB1C3|| &micro;L|| &micro;L|| &micro;L||
 +
|}
 +
 +
===Miniprep===
 +
<div class="experiment">
 +
<span class="author">tatsui</span>
 +
{|class="wikitable"
 +
!DNA||concentration[&micro;g/mL]||260/280||260/230
 +
|-
 +
|9/15 pSBiC3||111.7||1.79||1.36
 +
|-
 +
|9/15 spinach-DT||113.9||1.57||1.38
 +
|-
 +
|9/15 DT||77.0||2.05||1.92
 +
|}
 +
===PCR===
 +
<!-- ここから -->
 +
<div class="experiment">
 +
<span class="author">tatsui</span>
 +
{| class="wikitable"
 +
!9/14 aptamer12-P(VF)||Big Dye||5×Buffer||primer||MilliQ||total
 +
|-
 +
|1.5||0.5||1.75||0.5||5.75||10.5
 +
|}
 +
{| class="wikitable"
 +
!9/14 aptamer12-1M(VF)||Big Dye||5×Buffer||primer||MilliQ||total
 +
|-
 +
|1.7||0.5||1.75||0.5||6.05||10.5
 +
|}
 +
{| class="wikitable"
 +
!9/14 Fusion1 attenuator(VF)||Big Dye||5×Buffer||primer||MilliQ||total
 +
|-
 +
|2.3||0.5||1.75||0.5||5.45||10.5
 +
|}
 +
{| class="wikitable"
 +
!9/14 Fusion1 antisense(VF)||Big Dye||5×Buffer||primer||MilliQ||total
 +
|-
 +
|1.3||0.5||1.75||0.5||6.45||10.5
 +
|}
 +
{| class="wikitable"
 +
!9/13 Fusion3m2 attenuator(VF)||Big Dye||5×Buffer||primer||MilliQ||total
 +
|-
 +
|1.7||0.5||1.75||0.5||6.05||10.5
 +
|}
 +
{| class="wikitable"
 +
!9/13 Fusion6 antisense(VF)||Big Dye||5×Buffer||primer||MilliQ||total
 +
|-
 +
|1.6||0.5||1.75||0.5||6.15||10.5
 +
|}
 +
{| class="wikitable"
 +
!9/12 plux(VR)||Big Dye||5×Buffer||primer||MilliQ||total
 +
|-
 +
|2.4||0.5||1.75||0.5||5.35||10.5
 +
|}
 +
{| class="wikitable"
 +
!9/9 Ptet antisense(VF)||Big Dye||5×Buffer||primer||MilliQ||total
 +
|-
 +
|2.2||0.5||1.75||0.5||5.55||10.5
 +
|}
 +
{| class="wikitable"
 +
!9/12 Pcon-pT181 attenuator-DT(VF)||Big Dye||5×Buffer||primer||MilliQ||total
 +
|-
 +
|1.4||0.5||1.75||0.5||6.35||10.5
 +
|}
 +
{| class="wikitable"
 +
!9/12 Pcon-pT181 attenuator-DT(VR)||Big Dye||5×Buffer||primer||MilliQ||total
 +
|-
 +
|1.4||0.5||1.75||0.5||6.35||10.5
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|96&deg;C||96&deg;C||50&deg;C||60&deg;C||--
 +
|-
 +
|2min||10s||5s||4min||30cycles
|}
|}

Latest revision as of 02:00, 27 September 2013

Contents

Sep 16

Liquid Culture

No name

Samplemedium
pSB1C3+P-KaiBC①Plusgrow medium(+CP)
pSB1C3+P-KaiBC②Plusgrow medium(+CP)

Colony PCR

No name

Samplebase pair
pSB1C3+P-KaiBC①283
pSB1C3+P-KaiBC②283
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
2min30s30s18s30cycles

Gel Extraction

No name

LaneDNAEnzyme
11kb ladder
2pT181 attenuatorXbaI+PstI
3pT181 attenuatorXbaI+PstI
4pT181 attenuatorXbaI+PstI
6pSB1C3EcoRI+SpeI
7pSB1C3EcoRI+SpeI
8pSB1C3EcoRI+SpeI
10pSB4K5XbaI+PstI
11pSB4K5XbaI+PstI
12pSB4K5XbaI+PstI

Igku 9161.png
Igku 9161a.png

Nameconcentration[µg/mL]260/280260/230
pT181 attenuator(XbaI+PstI)8.21.620.11
pSB1C3(EcoRI+SpeI)49.51.480.18
pSB4K5(XbaI+PstI)18.11.730.57
DT(SpeI+PstI)70.41.361.02
aptamer12-1R-DT(SpeI+PstI)42.01.820.47
spinach-DT(SpeI+PstI)36.81.790.61

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
Dx-lux1136.71.461.28
RBS-laczα-DT237.61.881.70
pSB4K5148.41.551.09
Pcon-KBS-tetR-DT126.61.731.36
9/11Plac1160.01.781.28
9/15Plac2144.01.731.81
9/14Plac332.51.512.33
Ptet-RBS-laczα-DT231.31.841.57

PCR

No name

DNA9/6pT181 antisense psB1C3(1)KOD plus10x bufferdNTPMgSO4GG1_1C3_fwd primerGG2_1C3_rev primerMilliQtotal
400pg10.52.52.51.50.750.7515.525
800pg20.52.52.51.50.750.7514.525
1000pg2.50.52.52.51.50.750.751425
nega00.52.52.51.50.750.7516.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s30s30cycles
DNA9/6 12-1R aptamer-DTKOD plus10x bufferdNTPMgSO4GG1_1C3_fwd primerGG1_1C3_rev primerMilliQtotal
400pg10.52.52.51.50.750.7515.525
800pg20.52.52.51.50.750.7514.525
1000pg2.50.52.52.51.50.750.751425
NC00.52.52.51.50.750.7516.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s30s30cycles

Electrophoresis

Yoshida

LaneSample
1100bp ladder
2antisense 1000pg
3antisense 800pg
4antisense 400pg
5tet aptamer-DT 1000pg
6tet aptamer-DT 800pg
7tet aptamer-DT 400pg
8NC

Igku 9162.png

Restriction Enzyme Digestion

Tatsui

9/16 Pcon-RBS-tetR-DT(µL)EcoRI(µL)SpeI(µL)XbaI(µL)PstI(µL)Buffer(µL)BSA(µL)MilliQ(µL)total(µL)
2 cuts15.80011336.230
NC0.80000117.210
9/16 pSB4K5(µL)EcoRI(µL)SpeI(µL)XbaI(µL)PstI(µL)Buffer(µL)BSA(µL)MilliQ(µL)total(µL)
2 cuts13.50011338.530
NC0.70000117.310
8/22 pSB1C3(µL)EcoRI(µL)SpeI(µL)XbaI(µL)PstI(µL)Buffer(µL)BSA(µL)MilliQ(µL)total(µL)
2 cuts18.51001333.530
NC0.90000117.110
8/22 pSB1C3(µL)EcoRI(µL)SpeI(µL)XbaI(µL)PstI(µL)Buffer(µL)BSA(µL)MilliQ(µL)total(µL)
2 cuts18.51100333.530
NC0.90000117.110
8/21 pT181 attenuator2(µL)EcoRI(µL)SpeI(µL)XbaI(µL)PstI(µL)Buffer(µL)BSA(µL)MilliQ(µL)total(µL)
2 cuts7.411003314.630
NC0.40000117.610

Ligation

Tatsui

stateVectorInserterLigation High ver.2
experimentPcon-pT181antisense-spinach-DT(XbaI+PstI)5.0µg/mLpSB4K5(XbaI+PstI)18.1µg/mLxx µg/mL
experimentpC181 attenuator(XbaI+PstI)8.2µg/mLpSB1C3(XbaI+PstI)26.0µg/mLxx µg/mL

Transformation

tatsui

NameSampleCompetent CellsTotalPlate
Pcon+RBS-laczα-DT µL µL µL
Pcon-pT181 attenuator-DT+pSB1C3 µL µL µL
pT181 attenuator(EcoRI&SpeI)+pSB1C3 µL µL µL
Pcon-aptamer12_1R-DT(EcoRI&SpeI)+pSB1C3 µL µL µL
Pcon-spinach-DT(EcoRI&SpeI)+pSB1C3 µL µL µL
Pcon-RBS-tetR-DT(EcoRI&SpeI)+pSB1C3 µL µL µL
Pcon-pT181 antisense-spinach-DT(XbaI&PstI)+pSB1C3 µL µL µL

Miniprep

tatsui

DNAconcentration[µg/mL]260/280260/230
9/15 pSBiC3111.71.791.36
9/15 spinach-DT113.91.571.38
9/15 DT77.02.051.92

PCR

tatsui

9/14 aptamer12-P(VF)Big Dye5×BufferprimerMilliQtotal
1.50.51.750.55.7510.5
9/14 aptamer12-1M(VF)Big Dye5×BufferprimerMilliQtotal
1.70.51.750.56.0510.5
9/14 Fusion1 attenuator(VF)Big Dye5×BufferprimerMilliQtotal
2.30.51.750.55.4510.5
9/14 Fusion1 antisense(VF)Big Dye5×BufferprimerMilliQtotal
1.30.51.750.56.4510.5
9/13 Fusion3m2 attenuator(VF)Big Dye5×BufferprimerMilliQtotal
1.70.51.750.56.0510.5
9/13 Fusion6 antisense(VF)Big Dye5×BufferprimerMilliQtotal
1.60.51.750.56.1510.5
9/12 plux(VR)Big Dye5×BufferprimerMilliQtotal
2.40.51.750.55.3510.5
9/9 Ptet antisense(VF)Big Dye5×BufferprimerMilliQtotal
2.20.51.750.55.5510.5
9/12 Pcon-pT181 attenuator-DT(VF)Big Dye5×BufferprimerMilliQtotal
1.40.51.750.56.3510.5
9/12 Pcon-pT181 attenuator-DT(VR)Big Dye5×BufferprimerMilliQtotal
1.40.51.750.56.3510.5
PreDenatureDenatureAnnealingExtensioncycle
96°C96°C50°C60°C--
2min10s5s4min30cycles