Template:Kyoto/Notebook/Sep 27

From 2013.igem.org

(Difference between revisions)
(→RT PCR)
(→cDNA Synthesis)
 
(2 intermediate revisions not shown)
Line 432: Line 432:
|aptamer generator||0.348||0.408||0.550||0.865||||||||
|aptamer generator||0.348||0.408||0.550||0.865||||||||
|-
|-
-
|Pcon-spinach-DT||0.250||0.240||0.305||0.426||0.471||0.554||0.668||xx
+
|Pcon-spinach-DT||0.250||0.240||0.305||0.426||0.471||0.554||0.668||0
|-
|-
|GFP generator||0.389||0.420||0.516||0.884||||||||
|GFP generator||0.389||0.420||0.516||0.884||||||||
Line 438: Line 438:
|antisense-spinach||0.371||0.438||0.537||0.733||||||||
|antisense-spinach||0.371||0.438||0.537||0.733||||||||
|-
|-
-
|Spinach-DT||0.084||0.110||0.134||0.433||0.269||0.326||0.409||xx
+
|Spinach-DT||0.084||0.110||0.134||0.433||0.269||0.326||0.409||0.742
|-
|-
|}
|}
Line 461: Line 461:
<span class="author">Nakamoto</span>
<span class="author">Nakamoto</span>
{| class="wikitable"
{| class="wikitable"
-
!||9/27 Spinach-DT||9/23 Pcon-tetRaptamer-DT||9/23 Pcon-RBS-GFP-DT||9/23 Pcon-antisense-spinach-DT||9/23
+
!||9/27 Spinach-DT||9/23 Pcon-tetRaptamer-DT||9/23 Pcon-RBS-GFP-DT||9/23 Pcon-antisense-spinach-DT||9/23 Pcon-Spinach-DT
|-
|-
-
|RNA||1||1||1||1||x
+
|RNA||1||1||1||1||1
|-
|-
-
|gDNA wipeout||6||1.2||1.9||1.0||1.x
+
|gDNA wipeout||6||1.2||1.9||1.0||1.9
|-
|-
-
|buffer||||||1.9||1.0||1.x
+
|RNase-free water||0||4.8||4.1||5.0||4.1
|-
|-
-
|RNase-free water||0||4.8||4.1||5.0||4.x
+
|RT||0.5||0.5||0.5||0.5||0.5
-
|-
+
-
|RT||0.5||0.5||0.5||0.5||0.x
+
|-
|-
|RT Buffer||2||2||2||2||2
|RT Buffer||2||2||2||2||2

Latest revision as of 03:47, 28 September 2013

Contents

Sep 27

Gel Extraction

Kojima

LaneDNAEnzyme
11kbp ladder--
2pSB1C3EcoRI+SpeI
3
4
6Pcon-attenuator-aptmer-DTEcoRI+SpeI
7
8
10Pcon-attenuator-aptmer-DTEcoRI+SpeI
11
12

Igku 9272.jpg
Igku 9273.jpg

Nameconcentration[µg/mL]260/280260/230
Pcon-attenuator-DT(EcoRI+SpeI)6.81.980.43
pT181-attenuator(EcoRI+SpeI)7.51.840.06
pSB1C3(EcoRI+SpeI)17.71.970.71

Gel Extraction

No name

LaneDNAEnzyme
11kbp ladder--
3pSB4K5EcoRI+PstI
4pSB4K5EcoRI+PstI
5pSB4K5EcoRI+PstI
7pSB6A1EcoRI+PstI
8pSB6A1EcoRI+PstI
9pSB6A1EcoRI+PstI

Igku 9274.jpg
Igku 9275.jpg

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1pSB4K5EcoRIPstI
2pSB4K5--
31kbp ladder--
4Pcon-attenuatorEcoRISpeI
5Pcon-attenuator--


LaneSampleEnzyme1Enzyme2
1RBS-GFP-DTXbaIPstI
2RBS-GFP-DT--
3RBS-GFP-DTEcoRIXbaI
41kbp ladder--
5PtetEcoRISpeI
6Ptet--

Igku 0927 G5.jpg

Liquid Culture

No name

Samplemedium
Pcon-GFPAmp
RBS-GFP-DTCP
Pcon-attenuatorAmp
Pcon-attenuator-aptamer-DTAmp
Pcon-tetR-DTAmp

Ligation

No name

stateVectorInserterLigation High ver.2
experiment9/17 pSB1C3 (EcoRI+SpeI) 10.0ng/µL10µL9/26 pT181 attenuator(EcoRI+SpeI) 7.5ng/µL1.3µL3.5 µL
experiment9/25 DT (EcoRI+XbaI) 42.8ng/µL2.3µL9/22 Pcon-pT181 antisense(EcoRI+SpeI) 46.7ng/µL1.5µL1.9 µL
experiment9/27 pSB4K5(EcoRI+PstI) 12.9ng/µL7.8µL9/10 Pcon-attenuator(EcoRI+SpeI) 13.8ng/µL8.0µL3.5 µL
experiment9/27 pSB4K5(EcoRI+PstI) 12.9ng/µL7.8µL9/25 RBS-GFP-DT(XbaI+PstI) 13.9ng/µL12µL3.5 µL
experiment9/8 pSB4K5 (EcoRI+SpeI) 17.6ng/µL5.7 µL8/21 Pcon-GFP-DT(EcoRI+SpeI) 11ng/µL12.6µL3.5 µL

Liquid Culture

Hirano

Samplemedium
Pcon-spinach-DTplusgrow Amp
Pcon-tetRaptamer-DTplusgrow Amp　
Pcon-antisense-spinach-DTplusgrow Amp
spinach-DTplusgrow CP

RNA Extraction

Nakamoto

Nameconcentration[µg/mL]260/280260/230
9/26 Pcon-GFP-DT423.01.831.46
9/26 Pcon-GFP-DT K 298.71.821.50

Electrophoresis

No name

LaneSample
19/27 Pcon-GFP-DT(RNA)
21kb ladder
39/27 Pcon-GFP-DT K(RNA)

Igku 9271.jpg

Miniprep

Tatsui

DNA
Pcon-aptamer-DT
Pcon-spinach-DT
Pcon-antisense
Pcon-tetR-DT
Pcon-attenuator-DT
Pcon-antisense-spinach-DT

Transformation

Hirano

Name1Name2Sample1(µL)Sample2(µL)Competent Cells(µL)Total(µL)
Pcon-attenuator+RBS-GFP-DT(4K5)Pcon-attenuator-DT(1A2)213033
Pcon-attenuator+RBS-GFP-DT(4K5)Pcon-antisense-spinach-DT(1A2)213033
Pcon-attenuator+RBS-GFP-DT(4K5)Pcon-tetR aptamer-DT(1A2)213033
Pcon-RBS-GFP-DT(4K5)Pcon-attenuator-DT(1A2)213033
Pcon-RBS-GFP-DT(4K5)Pcon-antisense-spinach-DT(1A2)213033
Pcon-RBS-GFP-DT(4K5)Pcon-tetR aptamer-DT(1A2)213033
Pcon-attenuator+RBS-GFP-DT(4K5)--2--2022
Pcon-RBS-GFP-DT(4K5)--2--2022
attenuator(1C3)--2--2022

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
Pcon-RBS－GFP-DT137.61.951.78
RBS-GFP-DT77.02.061.96
Pcon-PT181 attenuator231.01.891.89
Pcon-antisense-Spinach-DT(2)130.01.561.62
Pcon-aptamer-DT218.81.921.72
Pcon-attenuator-DT180.71.951.78
Pcon-Spinach-DT177.61.961.06
Pcon-antisense-Spinach-DT(1)181.11.961.92
Pcon-antisense152.92.021.96
Pcon-RBS+ezR-DT164.11.931.91

Restriction Enzyme Digestion

No name

9/27 RBS-GFP-DTXbaIPstIBufferBSAMilliQtotal
2 cuts12µL1µL1µL3µL3µL20µL40µL
NC1.3µL0µL0µL1µL1µL6.7µL10µL
9/27 Pcon-tetR-DTEcoRIPstIBufferMilliQtotal
2 cuts12.2µL1µL1µL3µL12.8µL 30µL
NC0.6µL0µL0µL1µL8.4µL10µL

RNA Extraction

Nakamoto

Nameconcentration[µg/mL]260/280260/230
9/27 Pcon-RBS-GFP-DT686.61.991.34
9/27 Pcon-RBS-GFP-DT (+High salt)127.61.760.27

DNA sythesis

No name

genome DNADNA volumegenome DNA wipeout bufferRNase freewaterRTRT bufferPrimer Mixtotal
9/27 Pcon-RBS-GFP-DT0.8µL1µL5.2µL 0.5µL 2µL0.5µL 10µL
9/27 pcon-RBS-GFP-DT(+high salt)2.5µL1µL3.5µL 0.5µL 2µL 0.5µL 10µL
9/27 Pcon-RBS-GFP-DT(revised concentration) 0.5µL1µL5.5µL0.5µL2µL0.5µL10µL

Electrophoresis

No name

LaneSample
11kb ladder
2Pcon-RBS-GFP-DT
3 Pcon-RBS-GFP-DT

Igku 0927 E2.jpg

Liquid Culture

No name

Samplemedium
Pcon spinach DT plusgrow 2µL(Amp)
Pcon aptamer DT plusgrow 2µL(Amp)
Pcon antisense Spinach DT plusgrow 2µL(Amp)
Spinach DTplusgrow 4µL(CP)　

RT PCR

No name

genome DNAKOD plus10x buffer for KOD-plus-ver.22nM dNTPs25mM MgSO4F primerR primerMilliQtotal
0.512.52.51.50.750.7515.525
PreDenatureDenatureAnnealingExtensioncycle
　94°C　98°C　49.8°C　68°C--
2min　10sec　30sec　10sec　30

Electrophoresis

No name

LaneSample
1Pcon-GFP-DT(GFP)
2Pcon-GFP-DT(internal)
3Pcon-GFP-DT(HS)(GFP)
4Pcon-GFP-DT(internal)
LaneSample
1Pcon-GFP-DT(GFP)
2Pcon-GFP-DT(internal)
3100bp ladder

Igku 927E4.jpg

Digesting cut check

No name

LaneSample
1RBS-GFP-DT(XbaI&PstI)
2RBS-GFP-DT NC(XbaI&PstI)
31kbp ladder
4Pcon-RBS-tetR-DT(EcoRI&SpeI)
5Pcon-RBS-tetR-DT NC(EcoRI&SpeI)

Igku 927 E3.jpg

Gel Extraction

No name

LaneDNAEnzyme
11kbp ladder--
3RBS-GFP-DTXbal+PstI
4RBS-GFP-DTXbal+PstI
5RBS-GFP-DTXbal+PstI
71kbp ladder--
9Pcon-tetR-DTEcoRI+SpeI
10Pcon-tetR-DTEcoRI+SpeI
11Pcon-tetR-DTEcoRI+SpeI

Igku 0927 G7.jpg
Igku 0927 G8.jpg
Igku 0927 G9.jpg
Igku 0927 G10.jpg

RNA Extraction

Nakamoto

05min15min20min23min28min33min36min
aptamer generator0.3480.4080.5500.865
Pcon-spinach-DT0.2500.2400.3050.4260.4710.5540.6680
GFP generator0.3890.4200.5160.884
antisense-spinach0.3710.4380.5370.733
Spinach-DT0.0840.1100.1340.4330.2690.3260.4090.742
Nameconcentration[µg/mL]260/280260/230
Spinach-DT83.91.630.17
Pcon-tetRaptamer-DT425.12.100.64
Pcon-RBS-GFP-DT262.42.010.84
Pcon-antisense-Spinach-DT489.12.050.68
Pcon-Spinach-DT266.62.010.66

cDNA Synthesis

Nakamoto

9/27 Spinach-DT9/23 Pcon-tetRaptamer-DT9/23 Pcon-RBS-GFP-DT9/23 Pcon-antisense-spinach-DT9/23 Pcon-Spinach-DT
RNA11111
gDNA wipeout61.21.91.01.9
RNase-free water04.84.15.04.1
RT0.50.50.50.50.5
RT Buffer22222
Primer mix0.50.50.50.50.5
total10µL10µL10µL10µL10µL
Retrieved from "http://2013.igem.org/Template:Kyoto/Notebook/Sep_27"