Template:Kyoto/Notebook/Sep 1

From 2013.igem.org

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|8/31 tRNA Spinach(pSB1C3)||459
|8/31 tRNA Spinach(pSB1C3)||459
|-
|-
-
|8/31 pT181 antisense(2)(pSB1C3)(E+S)||415
+
|8/31 pT181 antisense(2)(pSB1C3)(EcoRI+SpeI)||415
|-
|-
-
|8/31 pT181 attenuator(2)(pSB1C3)(X+P)||601
+
|8/31 pT181 attenuator(2)(pSB1C3)(XbaI+PstI)||601
|-
|-
-
|8/31 pT181 antisense(2)(pSB1C3)(X+P)||415
+
|8/31 pT181 antisense(2)(pSB1C3)(XbaI+PstI)||415
|-
|-
|8/31 pT181 attenuator(2)-DT||754
|8/31 pT181 attenuator(2)-DT||754
Line 61: Line 61:
|5min||30s||30s||30min||30cycle
|5min||30s||30s||30min||30cycle
|}
|}
-
[[fig]]
+
[[File:Igku Sep1 ligation production PCR.jpg]]
</div>
</div>
 +
===Ligation===
===Ligation===
<div class="experiment">
<div class="experiment">
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!state||colspan="2"|Vector||colspan="2"|Inserter
!state||colspan="2"|Vector||colspan="2"|Inserter
|-
|-
-
|experiment||DT(E+S)||5.5||8/28 RBS-lysis1(E+S)2.4ng/&micro;L||2.4
+
|experiment||DT(EcoRI+SpeI)||5.5||8/28 RBS-lysis1(EcoRI+SpeI)2.4ng/&micro;L||2.4
|}
|}
1. Samples were evaporeted used evaporator into about 7 µL.<br>
1. Samples were evaporeted used evaporator into about 7 µL.<br>
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2 fold diluted OD 600 2.107
2 fold diluted OD 600 2.107
===Liquid Culture===
===Liquid Culture===
-
===Colony PCR===
+
<div class="experiment">
-
===Restriction Enzyme Digestion===
+
<span class="author">Hirano</span>
-
===Liquid Culture===
+
{| class="wikitable"
-
===Master Plate===
+
!Sample||medium
-
===Gel Extruction===
+
|-
-
===Colony PCR===
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|9/1 entA- Master Plate-(4)||SOB Medium(+Km) 　
-
===Master Plate===
+
|}
-
===Liquid Culture===
+
</div>

Latest revision as of 17:19, 25 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)(EcoRI+SpeI)415
8/31 pT181 attenuator(2)(pSB1C3)(XbaI+PstI)601
8/31 pT181 antisense(2)(pSB1C3)(XbaI+PstI)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

Igku Sep1 ligation production PCR.jpg

Ligation

Nakamoto

stateVectorInserter
experimentDT(EcoRI+SpeI)5.58/28 RBS-lysis1(EcoRI+SpeI)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°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)
RBS-lysis3-DTPlusgrow medium(+CP)


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
8/21 pSB1C3(1)309.21.691.72
8/21 pSB1C3(2)201.61.651.76

Kanamycin applicaton

Hirano

Add 20µL Kanamycin into 80µL MilliQ and apply it on a 9/1 M9 plate

Plating

Hirano

Apply entA-colony liquid culture (100µL) on M9(Km)plate and incubate 37°C

2 fold diluted OD 600 2.107

Liquid Culture

Hirano

Samplemedium
9/1 entA- Master Plate-(4)SOB Medium(+Km) 　
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