Template:Kyoto/Notebook/Sep 6

From 2013.igem.org

(Difference between revisions)
(→Electrophoresis)
(→Sep 6)
Line 35: Line 35:
|Plac-spinach-DT-(1~4)||659
|Plac-spinach-DT-(1~4)||659
|-
|-
-
|Pcon-PT181 antisense-(1~4)||468
+
|Pcon-pT181 antisense-(1~4)||468
|-
|-
-
|Ptet-PT181 antisense-(1~4)||604
+
|Ptet-pT181 antisense-(1~4)||604
|-
|-
-
|Pconst-PT181 attenuater-(1~4)||568
+
|Pconst-pT181 attenuater-(1~4)||568
|-
|-
|Pconst+RBS-tetR-DT-(1~4)||1121
|Pconst+RBS-tetR-DT-(1~4)||1121
Line 65: Line 65:
|9/4 RBS-Lysis1+DT3||Plusgrow medium(+Cp)
|9/4 RBS-Lysis1+DT3||Plusgrow medium(+Cp)
|-
|-
-
|9/4 PSB4K5||Plusgrow medium(+K)　
+
|9/4 pSB4K5||Plusgrow medium(+K)　
|}
|}
</div>
</div>
Line 91: Line 91:
|Plac-spinach-DT-(4)||-
|Plac-spinach-DT-(4)||-
|-
|-
-
|Pcon-PT181 antisense-(1)||-
+
|Pcon-pT181 antisense-(1)||-
|-
|-
-
|Pcon-PT181 antisense-(2)||-
+
|Pcon-pT181 antisense-(2)||-
|-
|-
-
|Pcon-PT181 antisense-(3)||-
+
|Pcon-pT181 antisense-(3)||-
|-
|-
-
|Pcon-PT181 antisense-(4)||-
+
|Pcon-pT181 antisense-(4)||-
|}
|}
Line 103: Line 103:
!|Sample||base pair
!|Sample||base pair
|-
|-
-
|Ptet-PT181 antisense-(1)||-
+
|Ptet-pT181 antisense-(1)||-
|-
|-
-
|Ptet-PT181 antisense-(2)||-
+
|Ptet-pT181 antisense-(2)||-
|-
|-
-
|Ptet-PT181 antisense-(3)||-
+
|Ptet-pT181 antisense-(3)||-
|-
|-
-
|Ptet-PT181 antisense-(4)||-
+
|Ptet-pT181 antisense-(4)||-
|-
|-
-
|Plac-PT181 attenuater-(1)||-
+
|Plac-pT181 attenuater-(1)||-
|-
|-
-
|Plac-PT181 attenuater-(2)||-
+
|Plac-pT181 attenuater-(2)||-
|-
|-
-
|Plac-PT181 attenuater-(3)||-
+
|Plac-pT181 attenuater-(3)||-
|-
|-
-
|Plac-PT181 attenuater-(4)||-
+
|Plac-pT181 attenuater-(4)||-
|}
|}
Line 126: Line 126:
!DNA||concentration [&micro;g/mL]||260/280||260/230
!DNA||concentration [&micro;g/mL]||260/280||260/230
|-
|-
-
|PT181 antisense-(1)||238.7||1.68||1.44
+
|pT181 antisense-(1)||238.7||1.68||1.44
|-
|-
-
|PT181 antisense-(2)||248.8||1.83||1.77
+
|pT181 antisense-(2)||248.8||1.83||1.77
|-
|-
-
|PT181 antisense-(3)||223.2||1.78||1.61
+
|pT181 antisense-(3)||223.2||1.78||1.61
|-
|-
-
|PT181 antisense-(4)||220.8||1.89||1.92
+
|pT181 antisense-(4)||220.8||1.89||1.92
|-
|-
|tetR aptamer-(1)||252.5||1.89||1.90
|tetR aptamer-(1)||252.5||1.89||1.90
Line 142: Line 142:
|121R aptamer-DT||251.7||1.92||2.01
|121R aptamer-DT||251.7||1.92||2.01
|-
|-
-
|PSB4K5||212.9||1.80||1.90
+
|pSB4K5||212.9||1.80||1.90
|-
|-
|RBS-lacZ&alpha;-DT||185.7||1.78||1.79
|RBS-lacZ&alpha;-DT||185.7||1.78||1.79
Line 172: Line 172:
{| class="wikitable"
{| class="wikitable"
-
! ||Pλ-luxI (86.4 µg/mL)||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
+
! ||P&lambda;-luxI (86.4 µg/mL)||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
|2 cuts||23.1&micro;L||1&micro;L||1&micro;L||0&micro;L||0&micro;L||3&micro;L||3&micro;L||1.9&micro;L||30&micro;L
|2 cuts||23.1&micro;L||1&micro;L||1&micro;L||0&micro;L||0&micro;L||3&micro;L||3&micro;L||1.9&micro;L||30&micro;L
Line 227: Line 227:
|1||100bp ladder||--
|1||100bp ladder||--
|-
|-
-
|2||rowspan="2"|Pλ-lux3||rowspan="2"|EcoRI & SpeI
+
|2||rowspan="2"|P&lambda;-lux3||rowspan="2"|EcoRI & SpeI
|-
|-
|3
|3
Line 244: Line 244:
|aptamer 12-1R-DT (XbaI&PstI)||15.3||1.93||0.70
|aptamer 12-1R-DT (XbaI&PstI)||15.3||1.93||0.70
|-
|-
-
|Pλ-lux3 (EcoRI&SpeI)||4.5||1.79||0.31
+
|P&lambda;-lux3 (EcoRI&SpeI)||4.5||1.79||0.31
|}
|}
</div>
</div>
Line 256: Line 256:
|9/5　Pconst-spinach-DT-(1)||Plusgrow medium(+Amp&CP)
|9/5　Pconst-spinach-DT-(1)||Plusgrow medium(+Amp&CP)
|-
|-
-
|9/5　Ptet-PT181 antisense-(1)||Plusgrow medium(+CP)
+
|9/5　Ptet-pT181 antisense-(1)||Plusgrow medium(+CP)
|-
|-
|9/5　Pconst+RBS-tetR-DT-(1)||Plusgrow medium(+Amp&CP)
|9/5　Pconst+RBS-tetR-DT-(1)||Plusgrow medium(+Amp&CP)
Line 262: Line 262:
|9/5　Ptet+RBS-lacZ&alpha;-DT-(1)||Plusgrow medium(+CP)
|9/5　Ptet+RBS-lacZ&alpha;-DT-(1)||Plusgrow medium(+CP)
|-
|-
-
|9/5　Pcon-PT181 attenuater-(1)||Plusgrow medium(+CP)
+
|9/5　Pcon-pT181 attenuater-(1)||Plusgrow medium(+CP)
|-
|-
|9/4　pT181 antisense-(1)||Plusgrow medium(+CP)
|9/4　pT181 antisense-(1)||Plusgrow medium(+CP)
Line 282: Line 282:
|9/5 Plac-attenuater-(5~12)||664
|9/5 Plac-attenuater-(5~12)||664
|-
|-
-
|9/5 PT181 antisense+Plac-(1)||-
+
|9/5 pT181 antisense+Plac-(1)||-
|}
|}
{| class="wikitable"
{| class="wikitable"

Revision as of 15:32, 24 September 2013

Contents

Sep 6

Electrophoresis

Hirano

LaneSample
1100bp ladder
29/4 RBS-lysis3-DT -3
39/4 RBS-lysis3-DT -4
49/4 RBS-lysis3-DT -5
59/4 RBS-lysis1-DT -3
69/4 Ptrc-KaiC -1
79/4 pSB4K5 -1
8100bp ladder

File:Igku xxxxxx.xxx

Colony PCR

No name

Samplebase pair
Pconst-spinach-DT-(1~4)563
Plac-spinach-DT-(1~4)659
Pcon-pT181 antisense-(1~4)468
Ptet-pT181 antisense-(1~4)604
Pconst-pT181 attenuater-(1~4)568
Pconst+RBS-tetR-DT-(1~4)1121
Ptet+RBS-lacZα-DT-(1~4)1162
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s40s30cycles


Liquid Culture

No name

Samplemedium
9/4 RBS-Lysis3+DT3Plusgrow medium(+Cp)
9/4 RBS-Lysis1+DT3Plusgrow medium(+Cp)
9/4 pSB4K5Plusgrow medium(+K)　

Colony PCR

No name

Samplebase pair
Pconst-spinach-DT-(1)-
Pconst-spinach-DT-(2)-
Pconst-spinach-DT-(3)-
Pconst-spinach-DT-(4)-
Plac-spinach-DT-(1)-
Plac-spinach-DT-(2)-
Plac-spinach-DT-(3)-
Plac-spinach-DT-(4)-
Pcon-pT181 antisense-(1)-
Pcon-pT181 antisense-(2)-
Pcon-pT181 antisense-(3)-
Pcon-pT181 antisense-(4)-
Samplebase pair
Ptet-pT181 antisense-(1)-
Ptet-pT181 antisense-(2)-
Ptet-pT181 antisense-(3)-
Ptet-pT181 antisense-(4)-
Plac-pT181 attenuater-(1)-
Plac-pT181 attenuater-(2)-
Plac-pT181 attenuater-(3)-
Plac-pT181 attenuater-(4)-

Miniprep

Honda and Kojima

DNAconcentration [µg/mL]260/280260/230
pT181 antisense-(1)238.71.681.44
pT181 antisense-(2)248.81.831.77
pT181 antisense-(3)223.21.781.61
pT181 antisense-(4)220.81.891.92
tetR aptamer-(1)252.51.891.90
tetR aptamer-(2)211.21.901.99
DT181 attenuater102.21.982.17
121R aptamer-DT251.71.922.01
pSB4K5212.91.801.90
RBS-lacZα-DT185.71.781.79
RBS-lysis2-DT-(1)84.81.852.24

Restriction Enzyme Digestion

Tatsui

9/6 RBS-lysis2-DT (78.3 µg/mL)EcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts22µL0µL0µL1µL1µL3µL3µL0µL30µL
NC1.3µL0µL0µL0µL0µL1µL1µL6.7µL10µL
  • at 37°C, for 1h
aptamer-DT (251.7 µg/mL)EcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts0.4µL0µL0µL1µL1µL3µL3µL14.1µL30µL
NC0.4µL0µL0µL0µL0µL1µL1µL7.6µL10µL
  • at 37°C, for 1h
Pλ-luxI (86.4 µg/mL)EcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts23.1µL1µL1µL0µL0µL3µL3µL1.9µL30µL
NC1.2µL0µL0µL0µL0µL1µL1µL6.8µL10µL
  • at 37°C, for 1h

Colony PCR

Hirano

Samplebase pair
Pconst+RBS-tetR-DT-(1)1121
Pconst+RBS-tetR-DT-(2)1121
Pconst+RBS-tetR-DT-(3)1121
Pconst+RBS-tetR-DT-(4)1121
Ptet+RBS-lacZα-DT-(1)1162
Ptet+RBS-lacZα-DT-(2)1162
Ptet+RBS-lacZα-DT-(3)1162
Ptet+RBS-lacZα-DT-(4)1162

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder--
2RBS-lysis2-DTXbaI & PstI
3
4----
5aptamer 12-1R-DTXbaI & PstI
6
LaneDNAEnzyme
1100bp ladder--
2Pλ-lux3EcoRI & SpeI
3
4
5
6
Nameconcentration[µg/mL]260/280260/230
RBS-lysis2-DT (XbaI&PstI)7.11.880.07
aptamer 12-1R-DT (XbaI&PstI)15.31.930.70
Pλ-lux3 (EcoRI&SpeI)4.51.790.31

Liquid Culture

No name

Samplemedium
9/5　Pconst-spinach-DT-(1)Plusgrow medium(+Amp&CP)
9/5　Ptet-pT181 antisense-(1)Plusgrow medium(+CP)
9/5　Pconst+RBS-tetR-DT-(1)Plusgrow medium(+Amp&CP)
9/5　Ptet+RBS-lacZα-DT-(1)Plusgrow medium(+CP)
9/5　Pcon-pT181 attenuater-(1)Plusgrow medium(+CP)
9/4　pT181 antisense-(1)Plusgrow medium(+CP)
9/4　aptamer 12-1R-(1)Plusgrow medium(+CP)

Colony PCR

No name

Samplebase pair
9/5 Plac-spinach-DT-(5~12)563
9/5 Pcon-antisense-(5~12)468
9/5 Plac-attenuater-(5~12)664
9/5 pT181 antisense+Plac-(1)-
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s42s30cycles
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