Template:Kyoto/Notebook/Sep 8

From 2013.igem.org

(Difference between revisions)
(→Gel Extraction)
(→PCR)
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<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
+
!KaiA(plasmid)||KOD plus||10x buffer||dNTP||MgSO4||Primer(661-RBS-KaiA-fwd)||Primer(kaiA-662-rev-ver2)||MilliQ||total
|-
|-
-
| ||0.5||2.5||2.5||1.5||0.75||0.75|| ||25
+
|1||0.5||2.5||2.5||1.5||0.75||0.75||15.5||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||28s||30cycles
|}
|}
 +
{| class="wikitable"
 +
!KaiB(plasmid)||KOD plus||10x buffer||dNTP||MgSO4||Primer(662-RBS-KaiB-fwd-ver2)||Primer(kaiB-663-rev-ver2)||MilliQ||total
 +
|-
 +
|1||0.5||2.5||2.5||1.5||0.75||0.75||15.5||25
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||98&deg;C||57&deg;C||68&deg;C||--
 +
|-
 +
|2min||10s||30s||28s||30cycles
 +
|}
 +
{| class="wikitable"
 +
!DT(plasmid)||KOD plus||10x buffer||dNTP||MgSO4||Primer(664-DT-fwd-ver2)||Primer(DT-suffix-660-rev-ver2)||MilliQ||total
 +
|-
 +
|0.1||0.5||2.5||2.5||1.5||0.75||0.75||16.4||25
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||98&deg;C||57&deg;C||68&deg;C||--
 +
|-
 +
|2min||10s||30s||28s||30cycles
 +
|}
 +
{| class="wikitable"
 +
!P-KaiB(PCR prpduct)||KOD plus||10x buffer||dNTP||MgSO4||Primer(prefix-PkaiBC-fwd-ver2)||Primer(PkaiBC-suffix--662-rev-ver2)||MilliQ||total
 +
|-
 +
|0.7||0.5||2.5||2.5||1.5||0.75||0.75||15.8||25
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||98&deg;C||57&deg;C||68&deg;C||--
 +
|-
 +
|2min||10s||30s||28s||30cycles
 +
|}
 +
{| class="wikitable"
 +
!Plac(1)(plasmid)||KOD plus||10x buffer||dNTP||MgSO4||Primer(GG0-pSBIC3-fwd-ver2)||Primer(Plac-GG1-rev-ver2)||MilliQ||total
 +
|-
 +
|0.13||0.5||2.5||2.5||1.5||0.75||0.75||16.37||25
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||98&deg;C||57&deg;C||68&deg;C||--
 +
|-
 +
|2min||10s||30s||60s||30cycles
 +
|}
 +
{| class="wikitable"
 +
!KaiC(plasmid)||KOD plus||10x buffer||dNTP||MgSO4||Primer(GG3-RBS-KaiC-fwd-ver2)||Primer(KaiC-GG4-rev-ver2)||MilliQ||total
 +
|-
 +
|1||0.5||2.5||2.5||1.5||0.75||0.75||15.5||25
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||98&deg;C||57&deg;C||68&deg;C||--
 +
|-
 +
|2min||10s||30s||60s||30cycles
 +
|}
 +
===Gel Extraction===
===Gel Extraction===
<!-- こっから -->
<!-- こっから -->

Revision as of 05:23, 24 September 2013

Contents

Sep 8

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder
2RBS-lysis1-DTXbaI & PstI
3RBS-lysis1-DTXbaI & PstI
5RBS-lysis3-DTXbaI & PstI
6RBS-lysis3-DTXbaI & PstI

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

Nameconcentration[µg/mL]260/280260/230
RBS-lysis1-DT (XbaI & PstI)5.31.980.06
RBS-lysis3-DT (XbaI & PstI)5.31.980.06
LaneDNAEnzyme
1100bp ladder
3Pcon-pT181 attenuatorEcoRI & SpeI
4Pcon-pT181 attenuatorEcoRI & SpeI
6Spinach-DTXbaI & PstI
7Spinach-DTXbaI & PstI
9RBS-lacZα-DTXbaI & PstI
10RBS-lacZα-DTXbaI & PstI

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

Nameconcentration[µg/mL]260/280260/230
Pcon-pT181 attenuator (EcoRI & SpeI)5.21.820.36
Spinach-DT (XbaI & PstI)8.01.870.25
RBS-lacZα-DT (XbaI & PstI)10.31.710.54
LaneDNAEnzyme
11kbp ladder
2Ptet-pT181 antisenseEcoRI & PstI
3Ptet-pT181 antisenseEcoRI & PstI
5pSB4K5EcoRI & SpeI
6pSB4K5EcoRI & SpeI

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

Nameconcentration[µg/mL]260/280260/230
Ptet-pT181 antisense(EcoRI & PstI)30.61.841.16
pSB4K5 (EcoRI & SpeI)21.81.870.98
LaneDNAEnzyme
11kbp ladder
2Pcon-pT181 attenuatorSpeI & PstI
3Pcon-pT181 attenuatorSpeI & PstI
5PlacSpeI & PstI
6PlacSpeI & PstI
8pSB1C3EcoRI & SpeI
9pSB1C3EcoRI & SpeI
11pSB1C3XbaI & PstI
12pSB1C3XbaI & PstI

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

Nameconcentration[µg/mL]260/280260/230
Pcon-pT181 attenuator (SpeI & PstI)14.51.911.09
Plac (SpeI & PstI)11.61.741.19
pSB1C3 (EcoRI & SpeI)5.71.740.34
pSB1C3 (XbaI & PstI)5.62.000.37

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
Pcon-pT181 anti243.01.901.81
Pcon-Spinach-DT253.71.571.51

Restriction Enzyme Digestion

じっけんしゃ

9/8 Pcon-pT181 antiESXPBSABufferMilliQtotal
2 cuts8.2µL 0µL 1µL 0µL 1µL 3µL 3µL 13.8µL 30µL
NC 0.4µL 0µL 0µL 0µL 0µL 1µL 1µL 7.6µL 10µL
9/8 Pcon-spin-DTESXPBSABufferMilliQtotal
2 cuts7.9µL 1µL 1µL 0µL 0µL 3µL 3µL14.1µL 30µL
NC 0.4µL 0µL 0µL 0µL 0µL 1µL 1µL7.6µL 10µL
8/21 F1 atteESXPBSABufferMilliQtotal
2 cuts 6.5µL 0µL 0µL 1µL 1µL 3µL 3µL15.5µL30µL
NC 0.3µL0µL0µL 0µL 0µL1µL1µL7.7µL10µL
8/21 F3m2 atteESXPBSABufferMilliQtotal
2 cuts6.0µL0µL0µL1µL1µL3µL3µL16µL30µL
NC 0.3µL0µL0µL 0µL 0µL1µL1µL7.7µL10µL
8/21 F1 antiESXPBSABufferMilliQtotal
2 cuts6.7µL0µL0µL1µL1µL3µL3µL15.3µL30µL
NC 0.3µL0µL0µL 0µL 0µL1µL1µL7.7µL10µL
8/21 F6 antiESXPBSABufferMilliQtotal
2 cuts6.0µL0µL0µL1µL1µL3µL3µL16µL30µL
NC 0.3µL0µL0µL 0µL 0µL1µL1µL7.7µL10µL
8/21 tetR aptamer 12_1MESXPBSABufferMilliQtotal
2 cuts5.3µL1µL1µL0µL0µL3µL3µL16.7µL30µL
NC 0.3µL0µL0µL 0µL 0µL1µL1µL7.7µL10µL
8/21 tetR aptamer 12_PESXPBSABufferMilliQtotal
2 cuts13.3µL1µL1µL0µL0µL3µL3µL8.7µL30µL
NC0.7µL0µL0µL0µL0µL1µL1µL7.3µL10µL
9/4 spin-DTESXPBSABufferMilliQtotal
2 cuts5.4µL0µL0µL1µL1µL3µL3µL16.6µL30µL
NC0.3µL0µL0µL0µL0µL1µL1µL7.7µL10µL
9/7 RBS-lysis1-DTESXPBSABufferMilliQtotal
2 cuts10µL0µL0µL1µL1µL3µL3µL12µL30µL
NC0.5µL0µL0µL0µL0µL1µL1µL7.5µL10µL
9/6 RBS-lysis2-DTESXPBSABufferMilliQtotal
2 cuts13µL0µL0µL1µL1µL3µL3µL9µL30µL
NC0.7µL0µL0µL0µL0µL1µL1µL7.3µL10µL
9/7 RBS-lysis3-DTESXPBSABufferMilliQtotal
2 cuts10µL0µL0µL1µL1µL3µL3µL12µL30µL
NC0.5µL0µL0µL0µL0µL1µL1µL7.5µL10µL
8/20 J23100 RBS-lacZα-DT-(1)ESXPBSABufferMilliQtotal
2 cuts7µL0.5µL0.5µL0µL0µL3µL3µL16µL30µL
NC0.5µL0µL0µL0µL0µL1µL1µL7.5µL10µL
8/20 RBS-lacZα-DTESXPBSABufferMilliQtotal
2 cuts10µL0.5µL0.5µL0µL0µL3µL3µL13µL30µL
NC0.5µL0µL0µL0µL0µL1µL1µL7.5µL10µL

Transformation

じっけんしゃ

NameSample(µL)Competent Cells(µL)Total(µL)Plate
9/8 Pcont+aptamer 12-1R-DT11011Amp
9/8 Pcon+RBS-lacZα-DT11011Amp
9/8 Pcon-pT181 attenuater+aptamer 12-1R-DT11011Amp
9/8 Plac+RBS-lysis1-DT22022CP
9/8 Plac+RBS-lysis2-DT22022CP
9/8 Plac+aptamer 12-1R-DT22022CP
9/8 Plac+spinach22022CP
9/8 Plac+pT181 attenuater22022CP
9/8 Plux+RBS-lysis1-DT11011CP
9/8 Plux+RBS-lysis2-DT11011CP
9/8 Ptet+RBS-lacZα-DT11011CP
9/8 Ptet-pT181 anti+spinach-DT11011CP
9/8 Pcon-pT181 attenuater+DT11011CP

Electrophoresis

じっけんしゃ

LaneSampleEnzyme1Enzyme2
1100bp ladder
2Pcon-spin-DTES
3Pcon-spin-DT
4Fusion1 attenuatorXP
5Fusion1 attenuator
6Fusion3m2 attenuatorXP
7Fusion3m2 attenuator
8Fusion1 antiXP
9Fusion1 anti
10Fusion6 antiXP
11Fusion6 anti
12aptamer 12-1MXP
13aptamer 12-1M
14aptamer 12-PXP
15aptamer 12-P
16spinach-DTXP
LaneSampleEnzyme1Enzyme2
1spinach-DT
2J23100-RBS-lacZα-DTES
3J23100-RBS-lacZα-DT
4100bp ladder
5RBS-lacZα-DTES
6RBS-lacZα-DT
7RBS-lysis1-DTXP
8RBS-lysis1-DT
LaneSampleEnzyme1Enzyme2
1100bp ladder
2RBS-lysis2-DTXP
3RBS-lysis2-DT
4RBS-lysis3-DTXP
5RBS-lysis3-DT
6Pcon pT181 antiSP
7Pcon pT181 anti

Gel Extraction

じっけんしゃ

LaneDNAEnzyme
1100bp ladder
2Pcon-spin-DTE+S
3Pcon-spin-DTE+S
5Fusion 1 attenuatorX+P
6Fusion 1 attenuatorX+P
8Fusion3m2 attenuatorX+P
9Fusion3m2 attenuatorX+P
11Fusion 1 antiX+P
12Fusion 1 antiX+P

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

Namequantityconcentration[µg/mL]260/280260/230
Pcon-spin-DT(E+S) 10.51.860.39
Fusion 1 attenuator(X+P) 7.01.710.27
Fusion3m2 attenuator(X+P) 8.61.750.47
Fusion 1 anti(X+P) 7.61.870.43
LaneDNAEnzyme
1100bp ladder
2Fusion6 antiX+P
3Fusion6 antiX+P
5aptamer12_1MX+P
6aptamer12_1MX+P

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

Namequantityconcentration[µg/mL]260/280260/230
Fusion6 anti(X+P) 5.01.760.20
aptamer12_1M(X+P) 3.32.310.04
LaneDNAEnzyme
1100bp ladder
2aptamer12_PX+P
3aptamer12_PX+P
5spinach-DTE+S
6spinach-DTE+S

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

Namequantityconcentration[µg/mL]260/280260/230
aptamer12_P(X+P) 3.41.720.26
spinach-DT(E+S) 10.11.570.47
LaneDNAEnzyme
1100bp ladder
2J23100-RBS-laczα-DTE+S
3J23100-RBS-laczα-DTE+S
5RBS-laczα-DTE+S
6RBS-laczα-DTE+S
8RBS-lysis1-DTX+P
9RBS-lysis1-DTX+P
11RBS-lysis2-DTX+P
12RBS-lysis2-DTX+P

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

Namequantityconcentration[µg/mL]260/280260/230
Pcon-RBS-laczα-DT(E+S) 5.62.030.25
RBS-laczα-DT(E+S) 4.42.040.33
RBS-lysis1-DT(X+P) 5.92.220.53
RBS-lysis2-DT(X+P) 5.11.670.48
LaneDNAEnzyme
1100bp ladder
2RBS-lysis3-DTX+P
3RBS-lysis3-DTX+P
5Pcon-pT181 antiS+P
6Pcon-pT181 antiS+P

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

Namequantityconcentration[µg/mL]260/280260/230
RBS-lysis3-DT(X+P) 23.41.720.88
Pcon-pT181 anti(S+P) 31.31.860.92

Ligation

じっけんしゃ

stateVectorInserterLigation High ver.2
experimentnamexx µg/mLnamexx µg/mLxx µg/mL
NCnamexx µg/mLnamexx µg/mLxx µg/mL
stateVectorInserterLigation High ver.2
experimentnamexx µg/mLnamexx µg/mLxx µg/mL
NCnamexx µg/mLnamexx µg/mLxx µg/mL

PCR

No name

KaiA(plasmid)KOD plus10x bufferdNTPMgSO4Primer(661-RBS-KaiA-fwd)Primer(kaiA-662-rev-ver2)MilliQtotal
10.52.52.51.50.750.7515.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s28s30cycles
KaiB(plasmid)KOD plus10x bufferdNTPMgSO4Primer(662-RBS-KaiB-fwd-ver2)Primer(kaiB-663-rev-ver2)MilliQtotal
10.52.52.51.50.750.7515.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s28s30cycles
DT(plasmid)KOD plus10x bufferdNTPMgSO4Primer(664-DT-fwd-ver2)Primer(DT-suffix-660-rev-ver2)MilliQtotal
0.10.52.52.51.50.750.7516.425
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s28s30cycles
P-KaiB(PCR prpduct)KOD plus10x bufferdNTPMgSO4Primer(prefix-PkaiBC-fwd-ver2)Primer(PkaiBC-suffix--662-rev-ver2)MilliQtotal
0.70.52.52.51.50.750.7515.825
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s28s30cycles
Plac(1)(plasmid)KOD plus10x bufferdNTPMgSO4Primer(GG0-pSBIC3-fwd-ver2)Primer(Plac-GG1-rev-ver2)MilliQtotal
0.130.52.52.51.50.750.7516.3725
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s60s30cycles
KaiC(plasmid)KOD plus10x bufferdNTPMgSO4Primer(GG3-RBS-KaiC-fwd-ver2)Primer(KaiC-GG4-rev-ver2)MilliQtotal
10.52.52.51.50.750.7515.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s60s30cycles

Gel Extraction

じっけんしゃ

LaneDNAEnzyme
（＞ω・）b

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

Namequantityconcentration[µg/mL]260/280260/230
（´ω｀）
（ΦωΦ^）
LaneDNAEnzyme
（＞ω・）b

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

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