Template:Kyoto/Notebook/Aug 21

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Contents

Aug 21

Plusgrow Medium

Tatsui

  • material
  • 8/17 Plusgrow medium 50ml
  • 7/22 5000x　Ampicillin 10µl
  1. Measuring materials and putting them in a 50ml tube

Liquid culture

Tatsui

Samplemedium
8/20 tRNA-spinach -(1)8/21 Plusgrow medium(+Amp)
8/20 tRNA-spinach -(2)8/21 Plusgrow medium(+Amp)
8/20 TetR-aptamer 12_P(1076) -(1)8/21 Plusgrow medium(+Amp)
8/20 TetR-aptamer 12_P(1076) -(2)8/21 Plusgrow medium(+Amp)
8/20 TetR-aptamer 12_1R(113) -(1)8/21 Plusgrow medium(+Amp)
8/20 TetR-aptamer 12_1R(113) -(2)8/21 Plusgrow medium(+Amp)
8/20 TetR-aptamer 12_1M(105) -(1) 8/21 Plusgrow medium(+Amp)
8/20 TetR-aptamer 12_1M(105) -(2)8/21 Plusgrow medium(+Amp)
8/20 PT181 attenuator -(1)8/21 Plusgrow medium(+Amp)
8/20 PT181 attenuator -(2)8/21 Plusgrow medium(+Amp)
8/20 Fusion1 attenuator -(1) 8/21 Plusgrow medium(+Amp)
8/20 Fusion1 attenuator -(2) 8/21 Plusgrow medium(+Amp)
8/20 Fusion3m2 attenuator -(1) 8/21 Plusgrow medium(+Amp)
8/20 Fusion3m2 attenuator -(2) 8/21 Plusgrow medium(+Amp)
8/20 PT181 antisense -(1) 8/21 Plusgrow medium(+Amp)
8/20 PT181 antisense -(2) 8/21 Plusgrow medium(+Amp)
8/20 Fusion1 antisense -(1) 8/21 Plusgrow medium(+Amp)
8/20 Fusion1 antisense -(2) 8/21 Plusgrow medium(+Amp)
8/20 Fusion6 antisense -(1) 8/21 Plusgrow medium(+Amp)
8/20 Fusion6 antisense -(2) 8/21 Plusgrow medium(+Amp)
  • incubate 37°C 10hour

Gel Extraction

Nakamoto and TaTsui

LaneDNAEnzyme
11kbp ladder--
28/20 Ptet -(1)EcoRI & XbaI
3
58/20 RBS-tetR-DT -(2)EcoRI & SpeI
6
88/20 Pconst-RBS-GFP-DT -(1)EcoRI & SpeI
9
118/20 RBS-lysis3 -(1)EcoRI & SpeI
12

[fig bifore][fig after]

Nameconcentration[µg/mL]260/280260/230
Ptet (EcoRI & XbaI)211.550.78
RBS-tetR-DT (EcoRI & SpeI)121.450.62
Pconst-RBS-GFP-DT (EcoRI & SpeI)111.360.60
RBS-lysis3 (EcoRI & SpeI)101.260.50

Ristriction Enzyme Digestion

Kojima and Nakamoto

8/20 Ptet-(1) EcoRIXbaI10x BSA10x buffer MMilliQtotal
4.5113317.530
0.50.20117.310
0.500.2117.310
0.500117.510
8/20 Pconst-RBS-luxR-DT-(2) EcoRIXbaI10x BSA10x buffer MMilliQtotal
2.9113319.130
0.30.20117.510
0.300.2117.510
0.300117.710
8/17 DTEcoRIXbaI10x BSA10x buffer MMilliQtotal
5.3113316.730
0.50.20117.310
0.500.2117.310
0.500117.510
  • incubate 37°C 1h

Electrophoresis

Kojima and Nakamoto

LaneSampleEnzyme1Enzyme2
11kbp ladder----
28/20 Ptet -(1)EcoRIXbaI
38/20 Ptet -(1)EcoRI--
48/20 Ptet -(1)--XbaI
58/20 Ptet -(1)----
68/20 J23100-RBS-luxR-DT -(2)EcoRIXbaI
78/20 J23100-RBS-luxR-DT -(2)EcoRI--
88/20 J23100-RBS-luxR-DT -(2)--XbaI
98/20 J23100-RBS-luxR-DT -(2)----
108/20 DTEcoRIXbaI
118/20 DTEcoRI--
128/20 DT--XbaI
138/20 DT----
141kbp ladder----
158/20 Ptet -(1)EcoRIXbaI
  • The reason why lane 2 and 15 were same is that the well of lane 2 might be broken.

[fig]
Kojima

LaneSampleEnzyme1Enzyme2
11kbp ladder----
28/20 J23100-RBS-luxR-DT -(2)EcoRIXbaI
38/20 DTEcoRIXbaI

[fig]

Gel Extraction

Honda

LaneDNAEnzyme
11kb ladder--
28/21 J23100-RBS-luxR-DT 25µLE+X
38/21 J23100-RBS-luxR-DT 25µLE+X
4----
58/21 Ptet 25µLE+X
68/21 Ptet 25µLE+X

File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx

Electrophoresis

Honda

LaneSampleEcoRIXbaI
11kb ladder----
28/21 DT++
38/21 DT+--
48/21 DT--+
58/21 DT----

File:Igku xxxxxx.xxx

Ligation

No name

Name＋NC
8/21 Pcn-RBS-luxR-DT(+Amp)2.32.3
8/21 Pcn-RBS-GFP-DT1.70
Ligation High buffer22
MilliQ01.7
total66
Name-?-NC
8/18 Plux(+CP)1.11.1
8/19 RBS-GFP-DT50
Ligation High buffer3.13.1
MilliQ05
total9.29.2
Name-?-NC
8/18 RBS(+Amp)2.2--
8/19 lysis16.0--
Ligation High buffer4.1--
MilliQ0--
total12.3--
Name-?-NC
8/18 RBS(+Amp)2.22.2
8/19 lysis23.60
Ligation High buffer2.92.9
MilliQ03.6
total8.78.7

Transformation

Okazaki

NameSampleCompetent CellsTotalPlate
8/21 Pcon-RBS-GFP-DT+Pcon-RBS-GFP-DT1µL10µL11µLAmp
8/21 Pcon-RBS-GFP-DT NC1µL10µL11µLAmp
8/18 RBS+8/19 lysis11µL10µL11µLAmp
8/18 RBS+8/19 lysis21µL10µL11µLAmp
8/18 RBS NC1µL10µL11µLAmp
8/18 Plux+8/18 RBS-GFP-DT1µL10µL11µLCP
8/18 Plux+8/18 RBS-GFP-DT1µL10µL11µLCP
8/21 Ptet(pm)+8/20 RBS-tetR-DT(2)1µL10µL11µLCP
8/21 Ptet(pm)+8/20 RBS-tetR-DT(2) NC1µL10µL11µLCP
8/21 pSB1C31µL10µL11µLCP

LB Medium Plate

Gel Extraction

Okazaki

LaneDNAEnzyme
11kb ladder--
2----
38/21 DTEcoRI & XbaI
4

File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx

Miniprep

Tatsui, Kojima, and Nakamoto

DNAconcentration [µg/mL]260/280260/230
tRNA-spinach-(1)2341.642.14
tRNA-spinach-(2)4401.601.91
tetR-aptamer 12-p(1076)-(1)1501.612.03
tetR-aptamer 12-p(1076)-(2)3741.451.66
tetR-aptamer 12-1R(113)-(1)3041.682.21
tetR-aptamer 12-1R(113)-(2)3821.471.66
tetR-aptamer 12-1M(105)-(1)3741.682.20
tetR-aptamer 12-1M(105)-(2)2861.311.43
PT181 attenuator-(1)3301.662.11
PT181 attenuator-(2)2701.682.18
Fusion1 attenuator-(1)3061.672.20
Fusion1 attenuator-(2)2821.601.94
Fusion3m2 attenuator-(1)3321.682.25
Fusion3m2 attenuator-(2)3261.672.10
PT181 antisense-(1)1781.641.68
PT181 antisense-(2)1161.631.95
Fusion1 antisense-(1)3001.672.21
Fusion1 antisense-(2)1641.421.46
Fusion6 antisense-(1)3361.652.18
Fusion6 antisense-(2)1921.652.01
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