Template:Kyoto/Notebook/Sep 17

From 2013.igem.org

(Difference between revisions)
(→Liquid Culture)
(→Gel Extraction)
 
(36 intermediate revisions not shown)
Line 6: Line 6:
!Sample||medium
!Sample||medium
|-
|-
-
|Spi-DT||LB medium(+CP)  
+
|Spinach-DT||LB medium(+CP)  
|-
|-
-
|Pcon-Spi-DT,Pcon-pT181-anti-spi-DT||LB medium(+Amp)　
+
|Pcon-Spinach-DT||LB medium(+Amp)　
 +
|-
 +
|Pcon-pT181 antisense-Spinach-DT||LB medium(+Amp)　
|}
|}
</div>
</div>
 +
===PCR===
===PCR===
-
<!-- ここから -->
+
<div class="experiment">
 +
<span class="author">Hirano</span>
 +
{| class="wikitable"
 +
!Pcon-tetR aptamer-DT(VF)||BigDye||5x buffer||primer(VF)||MilliQ||total
 +
|-
 +
|1.3||0.5||1.75||0.5||0.45||10.5
 +
|}
 +
{| class="wikitable"
 +
!Pcon-tetR aptamer-DT(VR)||BigDye||5x buffer||primer(VR)||MilliQ||total
 +
|-
 +
|1.3||0.5||1.75||0.5||0.45||10.5
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|96 &deg;C||96 &deg;C||50 &deg;C||60 &deg;C||--
 +
|-
 +
|2 min||10 sec||5 sec||4 min||30 cycles
 +
|}
 +
</div>
 +
 
 +
===Electrophoresis===
<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
+
!Lane||Sample||Enzyme1||Enzyme2
 +
|-
 +
|1||1kbp ladder||--||--
 +
|-
 +
|2||Pcon-RBS-tetR-DT||XbaI||PstI
 +
|-
 +
|3||Pcon-RBS-tetR-DT||--||--
 +
|-
 +
|4||pSB4K5||XbaI||PstI
 +
|-
 +
|5||pSB4K5||--||--
 +
|-
 +
|6||pSB1C3||EcoRI||SpeI
 +
|-
 +
|7||pSB1C3||EcoRI||PstI
 +
|-
 +
|8||pSB1C3||--||--
 +
|-
 +
|9||pT181 attenuator||EcoRI||SpeI
 +
|-
 +
|10||pT181 attenuator||--||--
 +
|-
 +
|11||1kbp ladder||--||--
 +
|}
 +
[[File:igku_9171.jpg]]<br>
 +
</div>
 +
 
 +
===Ligation===
 +
<div class="experiment">
 +
<span class="author">No Name</span>
 +
{| class="wikitable"
 +
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
 +
|-
 +
|experiment||8/28 Plux (SpeI & PstI)||5.0 &micro;L||9/13 RBS-lysis1-DT (XbaI & PstI)||3.5 &micro;L||3.5 &micro;L
 +
|-
 +
|experiment||8/28 Plux (SpeI & PstI)||5.0 &micro;L||9/13 RBS-lysis2-DT (XbaI & PstI)||3.6 &micro;L||3.5 &micro;L
 +
|-
 +
|experiment||8/28 Plux (SpeI & PstI)||11.0 &micro;L||9/13 RBS-lysis3-DT (XbaI & PstI)||9.5 &micro;L||3.5 &micro;L
 +
|-
 +
|experiment||9/14 Plac (SpeI & PstI)||2.4 &micro;L||9/16 pT181 attenuator-DT (XbaI & PstI)||4.8 &micro;L||3.5 &micro;L
 +
|-
 +
|experiment||9/14 Plac (SpeI & PstI)||2.4 &micro;L||9/3 pT181 antisense (XbaI & PstI)||2.0 &micro;L||2.2 &micro;L
 +
|-
 +
|experiment||9/14 Plac (SpeI & PstI)||2.4 &micro;L||9/10 aptamer 12_1R-DT (XbaI & PstI)||1.8 &micro;L||2.1 &micro;L
 +
|-
 +
|experiment||9/13 pSB1C3 (XbaI & PstI)||2.0 &micro;L||9/15 Pcon-PT181 antisense-Spinach-DT (XbaI & PstI)||12.0 &micro;L||3.5 &micro;L
 +
|-
 +
|experiment||9/16 pSB1C3 (EcoRI & SpeI)||1.0 &micro;L||9/15 Pcon-Spinach-DT (EcoRI & SpeI)||4.1 &micro;L||2.5 &micro;L
 +
|-
 +
|experiment||9/16 pSB1C3 (EcoRI & SpeI)||1.0 &micro;L||9/15 Pcon-RBS-tetR-DT (EcoRI & SpeI)||2.0 &micro;L||1.5 &micro;L
 +
|-
 +
|experiment||9/16 pSB1C3 (EcoRI & SpeI)||1.0 &micro;L||9/15 Pcon-PT181 attenuator-DT (EcoRI & SpeI)||2.4 &micro;L||1.7 &micro;L
 +
|-
 +
|experiment||9/16 pSB1C3 (EcoRI & SpeI)||1.0 &micro;L||9/15 Pcon-aptamer 12_1R-DT (EcoRI & SpeI)||2.0 &micro;L||1.5 &micro;L
 +
|-
 +
|experiment||9/13 pSB1C3 (XbaI & PstI)||2.0 &micro;L||9/16 Pcon-pT181-attenuator (XbaI & PstI)||4.4 &micro;L||3.2 &micro;L
 +
|}
 +
</div>
 +
 
 +
===Gel Extraction===
 +
<div class="experiment">
 +
<span class="author">No Name</span>
 +
{| class="wikitable"
 +
!Lane||DNA||Enzyme
 +
|-
 +
|1||1kbp ladder||--
 +
|-
 +
|2||rowspan="3"|pT181 attenuator -(2)||rowspan="3"|EcoRI & SpeI
 +
|-
 +
|3
 +
|-
 +
|4
 +
|}
 +
[[File:igku_9172.jpg]]<br>
 +
[[File:igku_9173.jpg]]<br>
 +
{| class="wikitable"
 +
!Name||concentration[&micro;g/mL]||260/280||260/230
 +
|-
 +
|pT181 attenuator (EcoI & SpeI)||9.2||1.15||0.12
 +
|}
 +
{| class="wikitable"
 +
!Lane||DNA||Enzyme
 +
|-
 +
|1||1kbp ladder||--
 +
|-
 +
|2||rowspan="3"|pSB1C3||rowspan="3"|EcoRI & SpeI
 +
|-
 +
|3
 +
|-
 +
|4
 +
|}
 +
[[File:igku_9174.jpg]]<br>
 +
[[File:igku_9175.jpg]]<br>
 +
{| class="wikitable"
 +
!Name||concentration[&micro;g/mL]||260/280||260/230
 +
|-
 +
|pSB1C3||10.0||1.84||0.40
 +
|}
 +
{| class="wikitable"
 +
!Lane||DNA||Enzyme
 +
|-
 +
|1||1kbp ladder||--
 +
|-
 +
|2||rowspan="3"|pSB4K5||rowspan="3"|XbaI & PstI
 +
|-
 +
|3
 +
|-
 +
|4
 +
|}
 +
[[File:igku_9176.jpg]]<br>
 +
[[File:igku_9177.jpg]]<br>
 +
{| class="wikitable"
 +
!Name||concentration[&micro;g/mL]||260/280||260/230
 +
|-
 +
|pSB4K5||7.6||1.81||0.32
 +
|}
 +
{| class="wikitable"
 +
!Lane||DNA||Enzyme
 +
|-
 +
|1||1kbp ladder||--
 +
|-
 +
|2||rowspan="3"|Pcon-RBS-tetR-DT||rowspan="3"|XbaI & PstI
 +
|-
 +
|3
 +
|-
 +
|4
 +
|}
 +
[[File:igku_9178.jpg]]<br>
 +
[[File:igku_9179.jpg]]<br>
 +
{| class="wikitable"
 +
!Name||concentration[&micro;g/mL]||260/280||260/230
 +
|-
 +
|Pcon-RBS-tetR-DT||8.0||2.40||1.14
 +
|}
 +
{| class="wikitable"
 +
!Lane||DNA||Enzyme
 +
|-
 +
|1||1kbp ladder||--
 +
|-
 +
|2||rowspan="3"|pSB1C3||rowspan="3"|EcoRI & PstI
 +
|-
 +
|3
 +
|-
 +
|4
 +
|}
 +
[[File:igku_91710.jpg]]<br>
 +
[[File:igku_91711.jpg]]<br>
 +
{| class="wikitable"
 +
!Name||concentration[&micro;g/mL]||260/280||260/230
 +
|-
 +
|pSB1C3||9.1||2.17||0.35
 +
|}
 +
</div>
 +
 
 +
===Mutation PCR===
 +
<div class="experiment">
 +
<span class="author">Kinose</span>
 +
{| class="wikitable"
 +
!pC KaiABC(100pg/&micro;L)||Prime STAR(2x)||Primer(KaiA-BsaI MuT-Fwd)||Primer(KaiA-BsaI MuT-Rev)||MilliQ||total
 +
|-
 +
|1.0||25.0||1.0||1.0||22.0||50.0
 +
|-
 +
|1.5||25.0||1.0||1.0||21.5||50.0
 +
|-
 +
|2.0||25.0||1.0||1.0||21.0||50.0
|-
|-
-
| ||0.5||2.5||2.5||1.5||0.75||0.75|| ||25
 
|}
|}
{| 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||98 &deg;C||65 &deg;C||72 &deg;C||--
|-
|-
-
|（◎Д◎）　||　||　||　||
+
|5 min||10 s||15 s||40 s||30 cycles
|}
|}
</div>
</div>
-
<!-- ここまでをコピーしてね -->
 

Latest revision as of 05:57, 26 September 2013

Contents

Sep 17

Liquid Culture

Tatsui and Hirano

Samplemedium
Spinach-DTLB medium(+CP)
Pcon-Spinach-DTLB medium(+Amp)　
Pcon-pT181 antisense-Spinach-DTLB medium(+Amp)　

PCR

Hirano

Pcon-tetR aptamer-DT(VF)BigDye5x bufferprimer(VF)MilliQtotal
1.30.51.750.50.4510.5
Pcon-tetR aptamer-DT(VR)BigDye5x bufferprimer(VR)MilliQtotal
1.30.51.750.50.4510.5
PreDenatureDenatureAnnealingExtensioncycle
96 °C96 °C50 °C60 °C--
2 min10 sec5 sec4 min30 cycles

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
11kbp ladder----
2Pcon-RBS-tetR-DTXbaIPstI
3Pcon-RBS-tetR-DT----
4pSB4K5XbaIPstI
5pSB4K5----
6pSB1C3EcoRISpeI
7pSB1C3EcoRIPstI
8pSB1C3----
9pT181 attenuatorEcoRISpeI
10pT181 attenuator----
111kbp ladder----

Igku 9171.jpg

Ligation

No Name

stateVectorInserterLigation High ver.2
experiment8/28 Plux (SpeI & PstI)5.0 µL9/13 RBS-lysis1-DT (XbaI & PstI)3.5 µL3.5 µL
experiment8/28 Plux (SpeI & PstI)5.0 µL9/13 RBS-lysis2-DT (XbaI & PstI)3.6 µL3.5 µL
experiment8/28 Plux (SpeI & PstI)11.0 µL9/13 RBS-lysis3-DT (XbaI & PstI)9.5 µL3.5 µL
experiment9/14 Plac (SpeI & PstI)2.4 µL9/16 pT181 attenuator-DT (XbaI & PstI)4.8 µL3.5 µL
experiment9/14 Plac (SpeI & PstI)2.4 µL9/3 pT181 antisense (XbaI & PstI)2.0 µL2.2 µL
experiment9/14 Plac (SpeI & PstI)2.4 µL9/10 aptamer 12_1R-DT (XbaI & PstI)1.8 µL2.1 µL
experiment9/13 pSB1C3 (XbaI & PstI)2.0 µL9/15 Pcon-PT181 antisense-Spinach-DT (XbaI & PstI)12.0 µL3.5 µL
experiment9/16 pSB1C3 (EcoRI & SpeI)1.0 µL9/15 Pcon-Spinach-DT (EcoRI & SpeI)4.1 µL2.5 µL
experiment9/16 pSB1C3 (EcoRI & SpeI)1.0 µL9/15 Pcon-RBS-tetR-DT (EcoRI & SpeI)2.0 µL1.5 µL
experiment9/16 pSB1C3 (EcoRI & SpeI)1.0 µL9/15 Pcon-PT181 attenuator-DT (EcoRI & SpeI)2.4 µL1.7 µL
experiment9/16 pSB1C3 (EcoRI & SpeI)1.0 µL9/15 Pcon-aptamer 12_1R-DT (EcoRI & SpeI)2.0 µL1.5 µL
experiment9/13 pSB1C3 (XbaI & PstI)2.0 µL9/16 Pcon-pT181-attenuator (XbaI & PstI)4.4 µL3.2 µL

Gel Extraction

No Name

LaneDNAEnzyme
11kbp ladder--
2pT181 attenuator -(2)EcoRI & SpeI
3
4

Igku 9172.jpg
Igku 9173.jpg

Nameconcentration[µg/mL]260/280260/230
pT181 attenuator (EcoI & SpeI)9.21.150.12
LaneDNAEnzyme
11kbp ladder--
2pSB1C3EcoRI & SpeI
3
4

Igku 9174.jpg
Igku 9175.jpg

Nameconcentration[µg/mL]260/280260/230
pSB1C310.01.840.40
LaneDNAEnzyme
11kbp ladder--
2pSB4K5XbaI & PstI
3
4

Igku 9176.jpg
Igku 9177.jpg

Nameconcentration[µg/mL]260/280260/230
pSB4K57.61.810.32
LaneDNAEnzyme
11kbp ladder--
2Pcon-RBS-tetR-DTXbaI & PstI
3
4

Igku 9178.jpg
Igku 9179.jpg

Nameconcentration[µg/mL]260/280260/230
Pcon-RBS-tetR-DT8.02.401.14
LaneDNAEnzyme
11kbp ladder--
2pSB1C3EcoRI & PstI
3
4

Igku 91710.jpg
Igku 91711.jpg

Nameconcentration[µg/mL]260/280260/230
pSB1C39.12.170.35

Mutation PCR

Kinose

pC KaiABC(100pg/µL)Prime STAR(2x)Primer(KaiA-BsaI MuT-Fwd)Primer(KaiA-BsaI MuT-Rev)MilliQtotal
1.025.01.01.022.050.0
1.525.01.01.021.550.0
2.025.01.01.021.050.0
PreDenatureDenatureAnnealingExtensioncycle
96 °C98 °C65 °C72 °C--
5 min10 s15 s40 s30 cycles
Retrieved from "http://2013.igem.org/Template:Kyoto/Notebook/Sep_17"