Template:Kyoto/Notebook/Sep 16

From 2013.igem.org

(Difference between revisions)
(→Miniprep)
(→PCR)
 
(20 intermediate revisions not shown)
Line 13: Line 13:
===Colony PCR===
===Colony PCR===
-
<!-- ここから -->
 
<div class="experiment">
<div class="experiment">
<span class="author">No name</span>
<span class="author">No name</span>
Line 33: Line 32:
===Gel Extraction===
===Gel Extraction===
<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"
!Lane||DNA||Enzyme
!Lane||DNA||Enzyme
|-
|-
-
|1||1kb ladder
+
|1||1kb ladder||
|-
|-
-
|2||pT181 attenuator||X+P
+
|2||pT181 attenuator||XbaI+PstI
|-
|-
-
|3||pT181 attenuator||X+P
+
|3||pT181 attenuator||XbaI+PstI
|-
|-
-
|4||pT181 attenuator||X+P
+
|4||pT181 attenuator||XbaI+PstI
|-
|-
-
|6||pSB1C3||E+S
+
|6||pSB1C3||EcoRI+SpeI
|-
|-
-
|7||pSB1C3||E+S
+
|7||pSB1C3||EcoRI+SpeI
|-
|-
-
|8||pSB1C3||E+S
+
|8||pSB1C3||EcoRI+SpeI
|-
|-
-
|10||pSB4K5||X+P
+
|10||pSB4K5||XbaI+PstI
|-
|-
-
|11||pSB4K5||X+P
+
|11||pSB4K5||XbaI+PstI
|-
|-
-
|12||pSB4K5||X+P
+
|12||pSB4K5||XbaI+PstI
|-
|-
|}
|}
-
[[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
|-
|-
-
|pT181 attenuator(X+P)||8.2||1.62||0.11
+
|pT181 attenuator(XbaI+PstI)||8.2||1.62||0.11
|-
|-
-
|pSB1C3(E+S)||49.5||1.48||0.18
+
|pSB1C3(EcoRI+SpeI)||49.5||1.48||0.18
|-
|-
-
|pSB4K5(X+P)||18.1||1.73||0.57
+
|pSB4K5(XbaI+PstI)||18.1||1.73||0.57
|-
|-
-
|DT(S+P)||70.4||1.36||1.02
+
|DT(SpeI+PstI)||70.4||1.36||1.02
|-
|-
-
|aptamer12-1R-DT(S+P)||42.0||1.82||0.47
+
|aptamer12-1R-DT(SpeI+PstI)||42.0||1.82||0.47
|-
|-
-
|spinach-DT(S+P)||36.8||1.79||0.61
+
|spinach-DT(SpeI+PstI)||36.8||1.79||0.61
|-
|-
|}
|}
Line 95: Line 94:
|9/15Plac2||144.0||1.73||1.81
|9/15Plac2||144.0||1.73||1.81
|-
|-
-
|9/14Plac332.5||1.51||2.33
+
|9/14Plac||332.5||1.51||2.33
|-
|-
|Ptet-RBS-laczα-DT||231.3||1.84||1.57
|Ptet-RBS-laczα-DT||231.3||1.84||1.57
Line 102: Line 101:
===PCR===
===PCR===
-
<!-- ここから -->
 
<div class="experiment">
<div class="experiment">
<span class="author">No name</span>
<span class="author">No name</span>
{| class="wikitable"
{| class="wikitable"
-
!genome DNA||KOD plus||10x buffer||dNTP||MgSO4||SasA_fwd primer||SasA_rev primer||MilliQ||total
+
{| class="wikitable"
 +
!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
 +
|-
 +
|800pg||2||0.5||2.5||2.5||1.5||0.75||0.75||14.5||25
 +
|-
 +
|1000pg||2.5||0.5||2.5||2.5||1.5||0.75||0.75||14||25
|-
|-
-
| ||0.5||2.5||2.5||1.5||0.75||0.75|| ||25
+
|nega||0||0.5||2.5||2.5||1.5||0.75||0.75||16.5||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||57&deg;C||68&deg;C||--
|-
|-
-
|（◎Д◎）　||　||　||　||
+
|2min||10s||30s||30s||30cycles
|}
|}
 +
{| class="wikitable"
 +
!DNA||9/6 12-1R aptamer-DT||KOD plus||10x buffer||dNTP||MgSO4||GG1_1C3_fwd primer||GG1_1C3_rev primer||MilliQ||total
 +
|-
 +
|-
 +
|400pg||1||0.5||2.5||2.5||1.5||0.75||0.75||15.5||25
 +
|-
 +
|800pg||2||0.5||2.5||2.5||1.5||0.75||0.75||14.5||25
 +
|-
 +
|1000pg||2.5||0.5||2.5||2.5||1.5||0.75||0.75||14||25
 +
|-
 +
|NC||0||0.5||2.5||2.5||1.5||0.75||0.75||16.5||25
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||98&deg;C||57&deg;C||68&deg;C||--
 +
|-
 +
|2min||10s||30s||30s||30cycles
 +
|}
 +
===Electrophoresis===
===Electrophoresis===
-
 
-
<!-- こっから -->
 
<div class="experiment">
<div class="experiment">
-
<span class="author">じっけんしゃ</span>
+
<span class="author">Yoshida</span>
{| class="wikitable"
{| class="wikitable"
-
!Lane||Sample||Enzyme1||Enzyme2
+
!Lane||Sample
|-
|-
-
|（°Д°）|| || ||  
+
|1||100bp ladder
 +
|-
 +
|2||antisense 1000pg
 +
|-
 +
|3||antisense 800pg
 +
|-
 +
|4||antisense 400pg
 +
|-
 +
|5||tet aptamer-DT 1000pg
 +
|-
 +
|6||tet aptamer-DT 800pg
 +
|-
 +
|7||tet aptamer-DT 400pg
 +
|-
 +
|8||NC
|}
|}
-
[[File:igku_xxxxxx.xxx]]<br>
+
[[File:igku_9162.png]]<br>
 +
 
===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">Tatsui</span>
{| class="wikitable"
{| class="wikitable"
-
! ||DNA||restricion enzyme||restriction enzyme||Buffer||BSA||MilliQ||total
+
! ||9/16 Pcon-RBS-tetR-DT(&micro;L)||EcoRI(&micro;L)||SpeI(&micro;L)||XbaI(&micro;L)||PstI(&micro;L)||Buffer(&micro;L)||BSA(&micro;L)||MilliQ(&micro;L)||total(&micro;L)
|-
|-
-
|2 cuts||（°°） &micro;L|| &micro;L|| &micro;L|| &micro;L|| &micro;L|| &micro;L|| &micro;L
+
|2 cuts||15.8||0||0||1||1||3||3||6.2||30
|-
|-
-
|NC|| &micro;L|| &micro;L|| &micro;L|| &micro;L|| &micro;L|| &micro;L|| &micro;L
+
|NC||0.8||0||0||0||0||1||1||7.2||10
|}
|}
 +
{| class="wikitable"
 +
! ||9/16 pSB4K5(&micro;L)||EcoRI(&micro;L)||SpeI(&micro;L)||XbaI(&micro;L)||PstI(&micro;L)||Buffer(&micro;L)||BSA(&micro;L)||MilliQ(&micro;L)||total(&micro;L)
 +
|-
 +
|2 cuts||13.5||0||0||1||1||3||3||8.5||30
 +
|-
 +
|NC||0.7||0||0||0||0||1||1||7.3||10
 +
|}
 +
{| class="wikitable"
 +
! ||8/22 pSB1C3(&micro;L)||EcoRI(&micro;L)||SpeI(&micro;L)||XbaI(&micro;L)||PstI(&micro;L)||Buffer(&micro;L)||BSA(&micro;L)||MilliQ(&micro;L)||total(&micro;L)
 +
|-
 +
|2 cuts||18.5||1||0||0||1||3||3||3.5||30
 +
|-
 +
|NC||0.9||0||0||0||0||1||1||7.1||10
 +
|}
 +
{| class="wikitable"
 +
! ||8/22 pSB1C3(&micro;L)||EcoRI(&micro;L)||SpeI(&micro;L)||XbaI(&micro;L)||PstI(&micro;L)||Buffer(&micro;L)||BSA(&micro;L)||MilliQ(&micro;L)||total(&micro;L)
 +
|-
 +
|2 cuts||18.5||1||1||0||0||3||3||3.5||30
 +
|-
 +
|NC||0.9||0||0||0||0||1||1||7.1||10
 +
|}
 +
{| class="wikitable"
 +
! ||8/21 pT181 attenuator2(&micro;L)||EcoRI(&micro;L)||SpeI(&micro;L)||XbaI(&micro;L)||PstI(&micro;L)||Buffer(&micro;L)||BSA(&micro;L)||MilliQ(&micro;L)||total(&micro;L)
 +
|-
 +
|2 cuts||7.4||1||1||0||0||3||3||14.6||30
 +
|-
 +
|NC||0.4||0||0||0||0||1||1||7.6||10
 +
|}
 +
===Ligation===
===Ligation===
-
 
-
<!-- こっから -->
 
<div class="experiment">
<div class="experiment">
-
<span class="author">じっけんしゃ</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||name||xx &micro;g/mL||name||xx &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
|-
|-
-
|NC||name||xx &micro;g/mL||name||xx &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===
===PCR===
<!-- ここから -->
<!-- ここから -->
<div class="experiment">
<div class="experiment">
-
<span class="author">No name</span>
+
<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"
{| class="wikitable"
-
!genome DNA||KOD plus||10x buffer||dNTP||MgSO4||SasA_fwd primer||SasA_rev primer||MilliQ||total
+
!9/12 Pcon-pT181 attenuator-DT(VR)||Big Dye||5×Buffer||primer||MilliQ||total
|-
|-
-
| ||0.5||2.5||2.5||1.5||0.75||0.75|| ||25
+
|1.4||0.5||1.75||0.5||6.35||10.5
|}
|}
{| class="wikitable"
{| class="wikitable"
!PreDenature||Denature||Annealing||Extension||cycle
!PreDenature||Denature||Annealing||Extension||cycle
|-
|-
-
|　&deg;C||　&deg;C||　&deg;C||　&deg;C||--
+
|96&deg;C||96&deg;C||50&deg;C||60&deg;C||--
|-
|-
-
|（◎Д◎）　||　||　||　||
+
|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
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