Template:Kyoto/Notebook/Sep 7

From 2013.igem.org

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)