Template:Kyoto/Notebook/Sep 7

From 2013.igem.org

(Difference between revisions)
(→Ligation)
(→Electrophoresis)
 
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===Colony PCR===
===Colony PCR===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Sample
 +
|-
 +
|Plac-spinach-DT -13~20
 +
|-
 +
|Plac-pT181 attenuator -13~20
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94 &deg;C||94 &deg;C||55 &deg;C||68&deg;C||--
 +
|-
 +
|5 min||30 s||30 s||42 sec||30 cycles
 +
|}
 +
</div>
 +
===Ligation===
===Ligation===
<div class="experiment">
<div class="experiment">
Line 131: Line 149:
</div>
</div>
-
===Colony PCR===
+
===Electrophoresis===
<div class="experiment">
<div class="experiment">
-
<span class="author">No name</span>
+
<span class="author">Nakamoto</span>
{| class="wikitable"
{| class="wikitable"
-
!Sample||base pair
+
!Lane||Sample
|-
|-
-
|9/5 Plac-spinarch-DT-13~20||
+
|1||100bp ladder
|-
|-
-
|9/5 Plac-pT181 attenuator-13~20||
+
|2||Plac-Spinach-DT -13
 +
|-
 +
|3||Plac-Spinach-DT -14
 +
|-
 +
|4||Plac-Spinach-DT -15
 +
|-
 +
|5||Plac-Spinach-DT -16
 +
|-
 +
|6||Plac-Spinach-DT -17
 +
|-
 +
|7||Plac-Spinach-DT -18
 +
|-
 +
|8||Plac-Spinach-DT -19
 +
|-
 +
|9||Plac-Spinach-DT -20
 +
|-
 +
|10||Plac-pT181 attenuator -13
 +
|-
 +
|11||Plac-pT181 attenuator -14
 +
|-
 +
|12||100bp ladder
|}
|}
 +
[[File:igku_974.jpg]]<br>
{| class="wikitable"
{| class="wikitable"
-
!PreDenature||Denature||Annealing||Extension||cycle
+
!Lane||Sample
|-
|-
-
|94　&deg;C||94　&deg;C||55　&deg;C||60　&deg;C||--
+
|1||100bp ladder
|-
|-
-
|5min　||30s　||30s　||42s　||30cycle
+
|2||Plac-pT181 attenuator -15
 +
|-
 +
|3||Plac-pT181 attenuator -16
 +
|-
 +
|4||Plac-pT181 attenuator -17
 +
|-
 +
|5||Plac-pT181 attenuator -18
 +
|-
 +
|6||Plac-pT181 attenuator -19
 +
|-
 +
|7||Plac-pT181 attenuator -20
 +
|-
 +
|8||100bp ladder
|}
|}
 +
[[File:igku_975.jpg]]<br>
</div>
</div>
 +
===Transformation===
===Transformation===
<div class="experiment">
<div class="experiment">
Line 159: Line 212:
|Pbed/araC+RBS-luxI-DT||2&micro;L ||20&micro;L ||22&micro;L ||
|Pbed/araC+RBS-luxI-DT||2&micro;L ||20&micro;L ||22&micro;L ||
|-
|-
-
|Plac+pT181 antisence ||2&micro;L ||20&micro;L ||22&micro;L ||
+
|Plac+pT181 antisense ||2&micro;L ||20&micro;L ||22&micro;L ||
|-
|-
|Pcon+RBS-luxI-DT ||2&micro;L ||20&micro;L ||22&micro;L ||
|Pcon+RBS-luxI-DT ||2&micro;L ||20&micro;L ||22&micro;L ||
|}
|}
</div>
</div>
 +
===Restriction Enzyme Digestion===
===Restriction Enzyme Digestion===
<div class="experiment">
<div class="experiment">
Line 207: Line 261:
! ||DNA||XbaI||PstI||Buffer||BSA||MilliQ||total
! ||DNA||XbaI||PstI||Buffer||BSA||MilliQ||total
|-
|-
-
|9/5 Spin-DT (XbaI & PstI)||5.4 &micro;L||1 &micro;L||1 &micro;L||3 &micro;L||3 &micro;L||16.6 &micro;L||30 &micro;L
+
|9/5 Spinach-DT (XbaI & PstI)||5.4 &micro;L||1 &micro;L||1 &micro;L||3 &micro;L||3 &micro;L||16.6 &micro;L||30 &micro;L
|-
|-
|NC||0.3 &micro;L||0 &micro;L||0 &micro;L||1 &micro;L||1 &micro;L||7.7 &micro;L||10 &micro;L
|NC||0.3 &micro;L||0 &micro;L||0 &micro;L||1 &micro;L||1 &micro;L||7.7 &micro;L||10 &micro;L
Line 238: Line 292:
|-
|-
|NC||0.3 &micro;L||0 &micro;L||0 &micro;L||1 &micro;L||1 &micro;L||7.7 &micro;L||10 &micro;L
|NC||0.3 &micro;L||0 &micro;L||0 &micro;L||1 &micro;L||1 &micro;L||7.7 &micro;L||10 &micro;L
 +
|}
 +
</div>
 +
 +
===Electrophoresis===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Lane||Sample||Enzyme1||Enzyme2
 +
|-
 +
|1||100bp ladder||--||--
 +
|-
 +
|2||RBS-lysis1-DT (9/7)||XbaI||PstI
 +
|-
 +
|3||RBS-lysis1-DT (9/7)||--||--
 +
|-
 +
|4||RBS-lysis3-DT (9/7)||XbaI||PstI
 +
|-
 +
|5||RBS-lysis3-DT (9/7)||--||--
 +
|-
 +
|6||Pcon-pT181 attenuator||EcoRI||SpeI
 +
|-
 +
|7||Pcon-pT181 attenuator||--||--
 +
|-
 +
|8||9/5 Spinach-DT||XbaI||PstI
 +
|-
 +
|9||9/5 Spinach-DT||--||--
 +
|-
 +
|10||100bp ladder||--||--
 +
|-
 +
|11||100bp ladder||--||--
 +
|-
 +
|12||RBS-lacZ&alpha;-DT||XbaI||PstI
 +
|-
 +
|13||RBS-lacZ&alpha;-DT||--||--
 +
|}
 +
[[File:igku_971.jpg]]<br>
 +
{| class="wikitable"
 +
!Lane||Sample||Enzyme1||Enzyme2
 +
|-
 +
|1||100bp ladder||--||--
 +
|-
 +
|2||Ptet-antisense||SpeI||PstI
 +
|-
 +
|3||Plac (NC)||--||--
 +
|-
 +
|4||pSB4K5||EcoRI||SpeI
 +
|-
 +
|5||pSB4K5||--||--
 +
|-
 +
|6||Pcon-pT181 attenuator||SpeI||PstI
 +
|-
 +
|7||1kbp ladder||--||--
 +
|}
 +
[[File:igku_972.jpg]]<br>
 +
{| class="wikitable"
 +
!Lane||Sample||Enzyme1||Enzyme2
 +
|-
 +
|1||100bp ladder||--||--
 +
|-
 +
|2||Plac||SpeI||PstI
 +
|-
 +
|3||Ptet-antisense||--||--
 +
|-
 +
|4||pSB1C3||EcoRI||SpeI
 +
|-
 +
|5||pSB1C3||--||--
 +
|-
 +
|6||pSB1C3||XbaI||PstI
 +
|-
 +
|7||pSB1C3||--||--
 +
|-
 +
|8||1kbp ladder||--||--
 +
|}
 +
[[File:igku_973.jpg]]<br>
 +
</div>
 +
 +
===Liquid Culture===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Sample||medium
 +
|-
 +
|entA- strain||SOB medium (+Kan)
 +
|-
 +
|9/15 Pcon-pT181 antisense||Plusgrow (+Amp)
|}
|}
</div>
</div>

Latest revision as of 02:13, 27 September 2013

Contents

Sep 7

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
RBS-lysis1-DT195.3
RBS-lysis3-DT255.5 1.91 2.04
Ptet-pT18183.6 1.85 1.48
Pcon-RBS-tetR-DT134.3 1.94 2.20
Ptet-RBS-lacZα-DT67.7 1.92 1.79
Pcon-pT181 attenuator232.3 1.95 1.90
pSB4K5237.0 1.61 1.50
pT181 antisense(pSB1C3)256.4 1.63 1.78
apt12_1R(pSB1C3)230.0 1.98 2.28

Electrophoresis

No name

LaneSample
1100bp ladder
2Plac-Spinach-DT -5
3Plac-Spinach-DT -6
4Plac-Spinach-DT -7
5Plac-Spinach-DT -8
6Plac-Spinach-DT -9
7Plac-Spinach-DT -10
8100bp ladder
1100bp ladder
2Plac-Spinach-DT -11
3Plac-Spinach-DT -12
4Pcon-antisense -5
5Pcon-antisense -6
6Pcon-antisense -7
7Pcon-antisense -8
8100bp ladder

Igku Sep7electrophoresis1.jpg

LaneSample
1100bp ladder
2Pcon-antisense -9
3Pcon-antisense -10
4Pcon-antisense -11
5Pcon-antisense -12
6Plac-attenuator -5
7Plac-attenuator -6
8100bp ladder
1100bp ladder
2Plac-attenuator -7
3Plac-attenuator -8
4Plac-attenuator -9
5Plac-attenuator -10
6Plac-attenuator -11
7Plac-attenuator -12
8100bp ladder

Igku Sep7electrophoresis2.jpg

LaneSample
1100bp ladder
2Plac-pT181 antisense

Igku Sep7electrophoresis3.jpg

Colony PCR

No name

Sample
Plac-spinach-DT -13~20
Plac-pT181 attenuator -13~20
PreDenatureDenatureAnnealingExtensioncycle
94 °C94 °C55 °C68°C--
5 min30 s30 s42 sec30 cycles

Ligation

No Name

stateVectorInserterLigation High ver.2
experiment9/6 Plux (SpeI&PstI)2 µL9/7 RBS-lysis2-DT (EcoRI&XbaI) µL3.5 µL
experiment9/6 Plac (SpeI&PstI) µL9/7 RBS-lysis2-DT (EcoRI&XbaI) µL3.5 µL
experiment9/6 Pcon-RBS-luxR-DT (EcoRI&XbaI)1.9 µL9/6 Plux-RBS-GFP-DT(SpeI&PstI)2.2 µL3.5 µL
experiment9/6 Pbed/araC (SpeI&PstI)1.6 µL9/5 RBS-luxI-DT (EcoRI&XbaI)16 µL3.5 µL
experiment9/6 Plac (SpeI&PstI)1.5 µL9/5 pT181 antisense (EcoRI&XbaI)2.0 µL3.5 µL
experiment9/6 Pcon (SpeI&PstI)3.0 µL9/5 RBS-luxI-DT (EcoRI&XbaI)10 µL3.5 µL

Electrophoresis

Nakamoto

LaneSample
1100bp ladder
2Plac-Spinach-DT -13
3Plac-Spinach-DT -14
4Plac-Spinach-DT -15
5Plac-Spinach-DT -16
6Plac-Spinach-DT -17
7Plac-Spinach-DT -18
8Plac-Spinach-DT -19
9Plac-Spinach-DT -20
10Plac-pT181 attenuator -13
11Plac-pT181 attenuator -14
12100bp ladder

Igku 974.jpg

LaneSample
1100bp ladder
2Plac-pT181 attenuator -15
3Plac-pT181 attenuator -16
4Plac-pT181 attenuator -17
5Plac-pT181 attenuator -18
6Plac-pT181 attenuator -19
7Plac-pT181 attenuator -20
8100bp ladder

Igku 975.jpg

Transformation

Tatsui

NameSampleCompetent CellsTotalPlate
Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT2µL20µL22µL
Pbed/araC+RBS-luxI-DT2µL 20µL 22µL
Plac+pT181 antisense 2µL 20µL 22µL
Pcon+RBS-luxI-DT 2µL 20µL 22µL

Restriction Enzyme Digestion

No Name

DNAXbaIPstIBufferBSAMilliQtotal
9/7 RBS-lysis1-DT(XbaI & PstI)10 µL1 µL1 µL3 µL3 µL12 µL30 µL
NC0.5 µL0 µL0 µL1 µL1 µL7.5 µL10 µL
DNAXbaIPstIBufferBSAMilliQtotal
9/7 RBS-lysis3-DT (XbaI & PstI)7.8 µL1 µL1 µL3 µL3 µL14.2 µL30 µL
NC0.4 µL0 µL0 µL1 µL1 µL7.6 µL10 µL
DNASpeIPstIBufferBSAMilliQtotal
9/5 Plac (SpeI & PstI)6.0 µL1 µL1 µL3 µL3 µL16.0 µL30 µL
NC0.3 µL0 µL0 µL1 µL1 µL7.7 µL10 µL
DNAEcorISpeIBufferBSAMilliQtotal
9/7 pSB4K5 (EcoRI & SpeI)8.4 µL1 µL1 µL3 µL3 µL16.0 µL30 µL
NC0.4 µL0 µL0 µL1 µL1 µL7.7 µL10 µL
DNArestricion enzymerestriction enzymeBufferBSAMilliQtotal
Pcon-pT181 attenuator (SpeI & PstI)8.6 µL1 µL1 µL3 µL3 µL13.4 µL30 µL
Pcon-pT181 attenuator (EcorI & SpeI)8.6 µL1 µL1 µL3 µL3 µL13.4 µL30 µL
NC0.4 µL0 µL0 µL1 µL1 µL7.6 µL10 µL
DNAXbaIPstIBufferBSAMilliQtotal
9/5 Spinach-DT (XbaI & PstI)5.4 µL1 µL1 µL3 µL3 µL16.6 µL30 µL
NC0.3 µL0 µL0 µL1 µL1 µL7.7 µL10 µL
DNASpeIPstIBufferBSAMilliQtotal
Ptet-anticodone (SpeI & PstI)11 µL1 µL1 µL3 µL3 µL11 µL30 µL
NC0.5 µL0 µL0 µL1 µL1 µL7.5 µL10 µL
DNAXbaIPstIBufferBSAMilliQtotal
9/6 RBS-lacZα-DT (XbaI & PstI)11 µL1 µL1 µL3 µL3 µL11 µL30 µL
NC0.5 µL0 µL0 µL1 µL1 µL7.5 µL10 µL
DNAEcoRISpeIBufferBSAMilliQtotal
pSB1C3 (1) (EcoRI & SpeI)6.6 µL1 µL1 µL3 µL3 µL15.4 µL30 µL
NC0.3 µL0 µL0 µL1 µL1 µL7.7 µL10 µL
DNAXbaIPstIBufferBSAMilliQtotal
pSB1C3 (3) (XbaI & PstI)6.6 µL1 µL1 µL3 µL3 µL15.4 µL30 µL
NC0.3 µL0 µL0 µL1 µL1 µL7.7 µL10 µL

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2RBS-lysis1-DT (9/7)XbaIPstI
3RBS-lysis1-DT (9/7)----
4RBS-lysis3-DT (9/7)XbaIPstI
5RBS-lysis3-DT (9/7)----
6Pcon-pT181 attenuatorEcoRISpeI
7Pcon-pT181 attenuator----
89/5 Spinach-DTXbaIPstI
99/5 Spinach-DT----
10100bp ladder----
11100bp ladder----
12RBS-lacZα-DTXbaIPstI
13RBS-lacZα-DT----

Igku 971.jpg

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2Ptet-antisenseSpeIPstI
3Plac (NC)----
4pSB4K5EcoRISpeI
5pSB4K5----
6Pcon-pT181 attenuatorSpeIPstI
71kbp ladder----

Igku 972.jpg

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2PlacSpeIPstI
3Ptet-antisense----
4pSB1C3EcoRISpeI
5pSB1C3----
6pSB1C3XbaIPstI
7pSB1C3----
81kbp ladder----

Igku 973.jpg

Liquid Culture

No name

Samplemedium
entA- strainSOB medium (+Kan)
9/15 Pcon-pT181 antisensePlusgrow (+Amp)
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