Template:Kyoto/Notebook/Sep 1

From 2013.igem.org

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===Miniprep===
===Miniprep===
<div class="experiment">
<div class="experiment">
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<span class="author">Kojima and Nakamoto</span>
 
{|class="wikitable"  
{|class="wikitable"  
!DNA||concentration[&micro;g/mL]||260/280||260/230  
!DNA||concentration[&micro;g/mL]||260/280||260/230  

Revision as of 03:42, 23 September 2013

Contents

Sep 1

Transformation

No name

NameSampleCompetent CellsTotalPlate
8/31 tRNA-Spinach(pSB1C3)2µL20µL22µLCP
8/31 pT181 antisense(pSB1C3)2µL20µL22µLCP
8/31 pT181 attenuator(pSB1C3)2µL20µL22µLCP
8/31 pT181 antisense(pSB1C3)2µL20µL22µLCP
8/31 pT181 attenuator(2)-DT2µL20µL22µLCP
8/31 pT181 antisense-DT2µL20µL22µLCP
8/31 tRNA-Spniach-DT2µL20µL22µLCP
8/31 RBS-lysis2-DT2µL20µL22µLCP
8/31 Pconst-RBS-tetR-DT2µL20µL22µLAmp
8/31 Plac-RBS-lacZα-DT2µL20µL22µLCP
8/31 Ptet-RBS-lacZα-DT2µL20µL22µLCP
8/31 2µL20µL22µLCP

ligation production PCR

No name

Samplebase pair
8/31 tRNA Spinach(pSB1C3)459
8/31 pT181 antisense(2)(pSB1C3)(E+S)415
8/31 pT181 attenuator(2)(pSB1C3)(X+P)601
8/31 pT181 antisense(2)(pSB1C3)(X+P)415
8/31 pT181 attenuator(2)-DT754
8/31 pT181 antisense(2)-DT577
8/31 tRNA Spinach-DT596
8/22 pSB1C3(2)(controll)1383
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s30min30cycle

fig

Ligation

Nakamoto

stateVectorInserterLigation High ver.2
experimentDT(E+S)5.58/28 RBS-lysis1(E+S)2.4ng/µL2.4

1. Samples were evaporeted used evaporator into about 7 µL.
2. Add Ligation High 3.5&micro:L and incubate 16&dig:C 1hour.

Liquid Culture

Tatsui,Stephane,Nakamoto

Samplemedium
8/21 pSB1C3(BBa_J04450)(1)Plusgrow medium(+CP)
8/21 pSB1C3(BBa_J04450)(2)Plusgrow medium(+CP)
8/18 RBS-lysis3-DTPlusgrow medium(+CP)
  • at the same time made maste plate.
  • incubate 37&dig:C 8hour.

M9 Medium

No name 5xM9 liquid medium

volume100mL
NaHPO430g
KH2PO3g
Nacl0.25g
NH4Cl0.5g
3.75% agar solution400mL
1M MgSO40.5mL
2M Glucose2.8mL
1% Vitamin B10.5mL
1M CaCl20.05mL

1. mix 5xM9 medium and 3.75%Agar solution 400mL
2. Add 1M MgSO4, 2M Glucose and 1 VitaminB1 0.5mL
3. Add CaCl2 while shaking Erlenmeyer flask.

Miniprep

DNAconcentration[µg/mL]260/280260/230
8/30 Plux-RBS-GFP-DT-(1)124　1.77　1.87
8/30 Plux-RBS-GFP-DT-(2)213　1.75　1.52
8/30 Pbad/araC-RBS-RFP427　1.70　1.56
8/30 Pbad/araC-RBS-RFP6701.300.83
8/30 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1)3321.732.04
8/30 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(2)4781.742.09
8/30 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(3)1461.772.11
8/30 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(4)4541.752.19

Kanamycin applicaton

Plating

Liquid Culture

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