Template:Kyoto/Notebook/Sep 23

From 2013.igem.org

Contents

Sep 23

Transformation

No name

NameSampleCompetent CellsPlate
9/14 pSB1C3+PkaiBC2 µL20 µL--
9/14 pSB1C3+RpaB2 µL20 µL--
9/14 pkaiBC+Rbs-GFP-DT2 µL20 µL--

Gel Extraction

No Name

LaneDNAEnzyme
1 1kbp ladder
3-4 Pcon-RBS-tetR-PT

Igku Sep23 Gel Extraction1.jpg
Igku Sep23 Gel Extraction2.jpg

Nameconcentration[µg/mL]260/280260/230
Pcon-PT181 anti EcoRI+SpeI46.7 1.06 0.93
Pcon-RBS-tetR-DT EcoRI+XbaI119.3 0.99
RBS-8FP-DT EcoRI+XbaI87.7 1.00 0.98
ap42_1R-DT EcoRI+XbaI68.8 1.32 1.03
Pcon-RBS-tetR-DT 1-2 A203.0 1.66 1.79
Pcon-RBS-tetR-DT 3-2 A67.7 2.64 1.28
Pcon-RBS-GFP-DT 1-S A185.6 1.88 2.04
Pcon-RBS-GFP-DT 2-S A298.6 1.68 1.55
Pcon-tetR-DT E-2 A202.4 1.57 1.66


PCR

No name

genome DNAKOD plus10x bufferdNTPMgSO4SasA_fwd primerSasA_rev primerMilliQtotal
9/8 Pcon-pT181 anti 0.52.52.51.50.750.7515.5 25
8/20 Ptet(1) 0.52.52.51.50.750.7515.5 25
9/16 Plac 2 0.52.52.51.50.750.7515.5 25
PreDenatureDenatureAnnealingExtensioncycle
94 °C98 °C57 °C68 °C--
2min 10sec 30sec 20sec 30cycles

Ligation

Yoshida

stateVectorInserterLigation High ver.2
experiment9/21 Pcon-RBS-GFP-DT EcoRI+XbaI47.5 µg/mL9/15 Pcon-RBS-tetR-DT EcoRI+SpeI59 µg/mL2.3 µg/mL
experiment9/21 Pcon-RBS-GFP-DT EcoRI+XbaI47.5 µg/mL9/15 Pcon-apt12_1R-DT EcoRI+SpeI17 µg/mL2.4 µg/mL
experiment9/21 Pcon-RBS-GFRDT EcoRI+XbaI2.1 µg/mL9/15 Pcon-DT181 attenuator-DT21 µg/mL3.0 µg/mL
experiment9/22 apt 12_1R-DT EcoRI+XbaI67.8 µg/mL9/18 Pcon-DT181 attenuator EcoRI+SpeI5.7 µg/mL3.5 µg/mL
experiment9/22RBS-GFP-DT EcoRI+XbaI87.7 µg/mL9/18 Pcon-DT18 attenuator EcoRI+SpeI6.7 µg/mL3.5 µg/mL
experiment9/13 DT EcoRI+XbaI25.4 µg/mL9/22 Pcon-DT181 antisense EcoRI+SpeI46.8 µg/mL2.3 µg/mL


PCR

No name

genome DNAKOD plus10x bufferdNTPMgSO4SasA_fwd primerSasA_rev primerMilliQtotal
Pcon-attenuator-DT (E-1A) 0.52.52.51.50.750.7577.5 25
Pcon-attenuator-DT (2-1A) 0.52.52.51.50.750.7577.5 25
Pcon-antisense-spinach-DT (1-2A) 0.52.52.51.50.750.7577.5 25
Pcon-antisense-spinach-DT (E-1A) 0.52.52.51.50.750.7577.5 25
PreDenatureDenatureAnnealingExtensioncycle
94 °C98 °C57 °C68 °C--
2min 10s 30s 45s 30cycle


Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
9/22 Plac-DT181 attenuator-1204.0 2.00 1.95
9/22 Plac-DT181 attenuator-2195.3 1.84 1.62
9/22 Plac-DT181 antisense-1188.2 1.87 2.15
9/22 Plac-DT181 antisense-2165.0 1.95 2.66

Electrophoresis

Hirano

LaneSampleEnzyme1Enzyme2
1,6100bp ladder(Promega)----
2Pcon-attenuator-DT (E-1A)----
3Pcon-attenuator-DT (2-1A)----
4Pcon-antisense-spinach-DT (1-2A)----
5Pcon-antisense-spinach-DT (E-1A)----

Igku 9232.png

Gel Extraction

Hirano

Nameconcentration[µg/mL]260/280260/230
Pcon-pT181 attenuator-DT(E-1A)166.7 1.51 0.99
Pcon-pT181 attenuator-DT(2-1A)122.1 1.75 1.20
Pcon-pT181 antisense-spinach-DT(1-2A)99.1 1.86 1.81

Electrophoresis

Hirano

LaneSampleEnzyme1Enzyme2
1100bp ladder----
29/8 Pcon-pT181 antisense(E-1A)----
38/20 Ptet(1)(2-S)----
49/16 Plac2(E-1A)----

Igku 9231.png

Transformation

No Name

NameSampleCompetent CellsTotalPlate
pSB1C32µL20µL22µL


Ligation

No Name

stateVectorInserterLigation High ver.2
experiment9/19 Plac SpeI+PstI24.6 µg/mL9/18 ape12_1R-DT XbaI+PstI4.5 µg/mL2 µg/mL
experiment9/21 RBS-GFP-DT EcoRI+XbaI88.4 µg/mL9/15 Pcon-pT181 attenuator EcoRI+SpeI21 µg/mL2 µg/mL
experiment9/13 PSB1C3 XbaI+PstI26.0 µg/mL9/16 pT181 attenuator XbaI+PstI8.2 µg/mL2 µg/mL


Colony PCR

Tatsui

Samplebase pair
9/22 Pcon-PT181 attenuator12-1R-DT(1~8)--
9/22 Pcon-PT181 attenuator(1~4)--
9/20 Plac+aptamer12-1R-DT(1~4)--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s54s30cycle