Team:Kyoto/Notebook

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<div id="kyoto-tab-contents">
<div id="kyoto-tab-contents">
<div id="kyoto-tab-NoteFFE">
<div id="kyoto-tab-NoteFFE">
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{{Kyoto/Notebook/FlowerFairyEcoli}}
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<ul class="kyoto-post-it">
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<li>[[File:Kyoto_FlorigenNote.png|30px|link=#FlorigenNotebook]]</li>
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<li>[[File:Kyoto_SecretionNote.png|30px|link=#SecretionNotebook]]</li>
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</ul>
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=Caution=
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{|border=1
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-
|[[File:causion.gif|100px]]We devided our team into 2 groups in order to achieve Flower Fairy E.coli,[[#FlorigenNotebook|Florigen]] and [[#SecretionNotebook|Secretion]] group. Florigen aims to confirm expression of florigen in E.coli, activation of R9 peptide, and function of FT made by E.coli. Secretion group aims to make a secretion system without cell death and confirm function of tatABCD and the other proteins.
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|}
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<html><a id="FlorigenNotebook"></a></html>
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=[[File:Kyoto_FlorigenNotebook.png]]=
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<div class="_kyoto-note">
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==August 2==
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====Mutation of FT====
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-
<small>by Sato</small><br>
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-
FT gene has two BioBrick restriction enzyme sites, EcoR1 and Pst1 which is next to each other.<br>
+
-
So we tried to delete both at once by using two primers with mutation.<br>
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-
{|class="wikitable"
+
-
|+Inverse PCR
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!10xBufer!!2mM dNTPs!!primer fwd!!primer rev!!template!!polymerase!!MilliQ!!Total
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|-
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|5||5||1.5||1.5||0.5||1||35.5||50
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-
|}
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94℃ 2min, (98℃ 10sec, 68℃ 4min)x4cycles, 4℃ Hold
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-
 
+
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{|class="wikitable"
+
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|+Dpn1 Digestion
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!PCR product!!Dpn1
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|-
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|50||2
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|}  37℃,1h incubate
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{|class="wikitable"
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|+Self-ligation
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!product!!MilliQ!!Ligase!!T4 Kinase!!Total
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|-
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|2||7||5||1||15
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-
|}
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16℃, 1h incubate
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-
 
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{|class="wikitable"
+
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|+Transformation
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!competent cell!!DNA
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-
|-
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|20||2
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-
|}
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Cells were stored on ice for 30min. <br>
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After 42℃ 60sec heat shock, cells were stored on ice for 2min.<br>
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Then cells were precultureed at 37℃ for 1hr, plated to Kanamycin plate.
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<br>
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<br>
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+
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==August 13==
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====Liquid culture====
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FT at 37°C, for overnight.
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+
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==August 14==
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====Miniprep of FT====
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<small>by Sato, Takeuchi</small><br>
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The concentration was 81.5ng/uL<br><br>
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+
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====Restriction digestion and Electrophoresis====
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<small> by Sato</small><br>
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To check wheter mutation was succeed, we did restriction enzyme digestion.
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{|class="wikitable"
+
-
!DNA(FT,80ng/uL)!!10xBuferH!!EcoR1!!Pst1!!MilliQ!!Total
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|-
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|5||2||1||-||12||20
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|-
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|5||2||-||1||12||20
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|}
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37°C 2h incubate<br>
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We did electrophoresis of three FT plasmids, non-cutted, EcoR1 cutted, Pst1 cutted.<br>
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However, we couldn't get any bands (data not shown.)<br>
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<br>
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====Liquid culture====
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FT (4mL)
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+
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==August 15==
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====Miniprep of FT====
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<small> by Sato, Takeuchi, Hyungcheol</small><br>
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We couldn't get enough concentration of plasmids.<br><br>
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====Electrophoresis====
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[[File:IMG_2177.jpg|200px|thumb|right]]
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<small> by Sato</small><br>
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We retried electrophoresis of three samples same as yesterday.<br>
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However, we couldn't get any bands as well.<br><br><br><br><br><br><br><br>
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-
 
+
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==August 16==
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====Transformation====
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<small> by Takeuchi, Ota</small><br>
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{| class="wikitable"
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!Name||Well||Sample||Competent Cells||Total||Plate||Colony
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|-
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|FT||-||1 &micro;L||10||11||LB (Kan+)||&#xD7;
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|-
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|pSB1C3||1-3-A||1||10||11||LB (CP+)||&#x25CB;
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|-
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|I719005||1-15-N||1||10||11||LB (Amp+)||&#x25CB;
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|}
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==August 17==
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====Transformation====
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<small> by Takeuchi</small><br>
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{| class="wikitable"
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!Name||Well||Sample||Competent Cells||MilliQ||Total||Plate||Colony
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|-
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|FT||-||2||2||18||22||LB (Kan+)||&#xD7;
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|}
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We found that mutation of FT was not successful.
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==August 20==
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We decided to do PCR using FT specific primers before mutation.<br>
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====PCR of FT====
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[[File:IMG_2178.jpg|200px|thumb|right]]
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<small> by Sato</small><br>
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{|class="wikitable"
+
-
!10xBufer!!dNTPs!!MgSO4!!primer fwd!!primer rev!!template!!polymerase!!MilliQ!!Total
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-
|-
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|5||5||3||1||1||1(130ng/&micro;L)||1(KOD plus neo)||33||50
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-
|}
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94°C 2min,  (98°C 10sec, 68°C 15sec)x30cycles,  4°C Hold<br>
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After the ethanol precipitation, we diluted in 30&micro;L of MilliQ.<br>
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The concentration was 203ng/&micro;L.
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-
<br>
+
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<br>
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-
 
+
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====Electrophoresis====
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[[File:Electrophoresis0821.png|400px|right]]
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{| class="wikitable"
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!Lane!!Name!!length(bp)
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|-
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|1||1kb ladder||-
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|-
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|2||FT||600
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-
|}
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<br style="clear:both" />
+
-
 
+
-
==August 21==
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====Restriction digestion====
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-
<small> by Sato</small><br>
+
-
{|class="wikitable"
+
-
!DNA(FT,203ng/&micro;L)!!10xBuferM!!Xba11!!Pst1!!MilliQ!!Total
+
-
|-
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-
|10||4||1||1||24||40
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-
|}
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37°C, 5h incubate<br>
+
-
After the ethanol precipitation, we diluted in 30&micro;L of MilliQ.<br>
+
-
The concentration was 54,9ng/&micro;L.<br>
+
-
<br>
+
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====Ligation====
+
-
<small> by Sato</small><br>
+
-
{| class="wikitable"
+
-
!colspan="2"|Vector||colspan="2"|Insert||Ligation High Ver.2
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-
|-
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|pSB1C3||1||FT||10||5.5
+
-
|}
+
-
 
+
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====Liquid culture====
+
-
T7 promoter, pSB1C3 (4mL)
+
-
<br>
+
-
<br>
+
-
==August 22==
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-
====Miniprep====
+
-
<small> by Sato</small><br>
+
-
{| class="wikitable"
+
-
!T7 promoter||pSB1C3
+
-
|-
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-
|85.3ng/&micro;L||82.93ng/&micro;L
+
-
|}
+
-
<br>
+
-
 
+
-
==August 23==
+
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====Ethanol Precipitation<br>====
+
-
<small>diluted in 20µL 79.3ng/µL</small><br>
+
-
<br>
+
-
 
+
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==August 24==
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====Restriction enzyme processing====
+
-
[[File:IMG_2179.jpg|200px|thumb|right]]
+
-
{|class="wikitable"
+
-
!T7 promoter(85.3ng/µL)!!Spel!!Pstl!!buffer M!!MiliQ!!Total
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-
|-
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|10||1||1||2||6||20
+
-
|}
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->purifying column 33.4ng/µL(dissolution 40µL)<br>
+
-
{|class="wikitable"
+
-
!pSB1C3(82.9ng/µL)!!Xbal!!Spel!!buffer M!!MiliQ!!Total
+
-
|-
+
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|20||1||1||4||14||40
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-
|}
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->gene clean2 39.9ng/µL(dissolution 40µL)<br>
+
-
<br>
+
-
 
+
-
<br>
+
-
 
+
-
====Ligation====
+
-
{|class="wikitable"
+
-
!FT(600bp, 79.3ng/µL)!!pSB1C3(2000bp,39.9ng/µL)!!Ligation High Ver.2
+
-
|-
+
-
|3µL => 597fmol||2µL => 60fmol||2.5µL
+
-
|}
+
-
{|class="wikitable"
+
-
!FT(600bp, 79.3ng/µL)!!T7(2100bp,33.4ng/µL)!!Ligation High Ver.2
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-
|-
+
-
|2.4µL => 478fmol||2µL => 48fmol||2.2µL
+
-
|}
+
-
=> 16℃,1hr incubate
+
-
 
+
-
==August 27==
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-
====Colony PCR====
+
-
[[File:IMG_2180.jpg|200px|thumb|right]]
+
-
{|class="wikitable"
+
-
!2X Quick Tag!!VF2!!VR!!MiliQ!!Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
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Lane1: 1kb ladder<br>
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Lane2~16: FT(pSB1C3) about 800bp<br>
+
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====FT(TOPO) PCR(re)====
+
-
 
+
-
[[File:IMG_2181.jpg|200px|thumb|right]]
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-
{|class="wikitable"
+
-
!buffer!!dNTPs!!MgSO4!!primer f!!primer r!!Template(130ng/µL)!!KODplus neo!!MilliQ!!Total
+
-
|-
+
-
|5||5||3||1||1||1||1||33||50
+
-
|}
+
-
 
+
-
{| class="wikitable"
+
-
!94℃||98℃||68℃||cycles
+
-
|-
+
-
|2min||10sec||15sec||30
+
-
|}
+
-
<br>
+
-
====Liquid culture====
+
-
<small>by Nobeyama</small><br>
+
-
FT 4ml<br>
+
-
 
+
-
==August 28==
+
-
 
+
-
====Mutation of FT (re)====
+
-
[[File:IMG_2182.jpg|200px|thumb|right]]
+
-
inverse PCR<br>
+
-
{|class="wikitable"
+
-
!MilliQ!!buffer!!dNTP!!primer f!!primer r!!FT(130ng/µL)!!KODplus!!Total
+
-
|-
+
-
|35.5||5||5||1.5||1.5||0.5||1||50
+
-
|}
+
-
{| class="wikitable"
+
-
!94℃||98℃||68℃||cycles
+
-
|-
+
-
|2min||10sec||4min||18
+
-
|}<br>
+
-
Lane1: 1kb ladder<br>
+
-
Lane2: FT<br>
+
-
 
+
-
====Miniprep FT(TOPO)====
+
-
158ng/µL<br><br>
+
-
 
+
-
====Tranformation====
+
-
competent cell: 20<br>
+
-
BBa.I746902  : 2<br>
+
-
(plate 316f)<br>
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(GFP generator of pBAD/araC-mut3 GFP:6His-DT)<br>
+
-
 
+
-
==August 29==
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====Mutaion of FT(re;re)====
+
-
====Inverse PCR====
+
-
first
+
-
{|class="wikitable"
+
-
!MilliQ!!buffer!!dNTP!!primer f!!primer r!!FT(130ng/µL)!!KODplus!!Total
+
-
|-
+
-
|35.5||5||5||1.5||1.5||0.5||1||50
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-
|}
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second
+
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{|class="wikitable"
+
-
!MilliQ!!buffer!!dNTP!!primer f!!primer r!!FT(52ng/µL)!!KODplus!!Total
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|-
+
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|35||5||5||1.5||1.5||1||1||50
+
-
|}
+
-
{| class="wikitable"
+
-
!94℃||98℃||68℃||cycles
+
-
|-
+
-
|2min||10sec||4min||30
+
-
|}<br>
+
-
Lane1: 1kb ladder<br>
+
-
Lane2: first Template 130ng/µL<br>
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-
Lane3: second Template 53ng/µL<br>
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-
[[File:IMG_2183.jpg|200px|thumb|right]]
+
-
{| class="wikitable"
+
-
!first sample||Dpnl||total
+
-
|-
+
-
|45µL||2µL||47µL
+
-
|}
+
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in 37℃, 1 hour<br>
+
-
 
+
-
====Self-Ligation====
+
-
 
+
-
{| class="wikitable"
+
-
!PCR products||MilliQ||Ligation High||T4 kinase||total
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-
|-
+
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|2µL||7µL||5µL||1µL||15µL
+
-
|}<br>
+
-
in 16℃, 1.5hour incubate<br>
+
-
Transformation<br>
+
-
competent cell: 20<br>
+
-
DNA          : 2<br><br>
+
-
Liquid culture(I746902): 3mL
+
-
 
+
-
==August 30==
+
-
====Liquid culture(FT) 4mL x2====
+
-
 
+
-
==August 31==
+
-
====Miniprep(FT)====
+
-
 
+
-
(1) 64.9ng/µL<br>
+
-
(2) 52.6ng/µL<br>
+
-
 
+
-
====Restriction enzyme processing (Mutation checking)====
+
-
{| class="wikitable"
+
-
!FT(52.6ng/µL)||bufferH||E.coli||Pst1||MilliQ||total
+
-
|-
+
-
|1µL||5µL||0.5µL||0.5µL||3.5µL||10µL
+
-
|}<br>
+
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in 37℃,1.5hour<br>
+
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====PCR(RBS primer)====
+
-
 
+
-
{|class="wikitable"
+
-
!buffer for KODplus neo!!dNTPs!!MgSO4!!primer f!!primer r!!Template(52.6ng/µL)!!KODplus neo!!MilliQ!!Total
+
-
|-
+
-
|5||5||3||1||1||1||1||33||50
+
-
|}<br>
+
-
[[File:IMG_2184.jpg|200px|thumb|right]]
+
-
{| class="wikitable"
+
-
!94℃||98℃||68℃||cycles
+
-
|-
+
-
|2min||10sec||15sec||30
+
-
|}<br>
+
-
 
+
-
Lane1: 1kb ladder<br>
+
-
Lane2: FT<br>
+
-
Lane3: FT(E.coli)<br>
+
-
Lane4: FT(Pst1)<br>
+
-
Lane5: FT PCR<br><br>
+
-
 
+
-
====PCR(re)====
+
-
 
+
-
{|class="wikitable"
+
-
!buffer!!dNTPs!!MgSO4!!primer f!!primer r!!Template!!KOD neo!!MilliQ!!Total
+
-
|-
+
-
|5||5||3||1||1||1||1||33||50
+
-
|}<br>
+
-
[[File:IMG_2185.jpg|200px|thumb|right]]
+
-
{| class="wikitable"
+
-
!94℃||98℃||68℃||cycles
+
-
|-
+
-
|2min||10sec||15sec||35
+
-
|}<br>
+
-
 
+
-
Lane1: 1kb ladder<br>
+
-
Lane2: FT<br>
+
-
 
+
-
====PCR(re)====
+
-
 
+
-
{|class="wikitable"
+
-
!buffer!!dNTPs!!MgSO4!!primer f!!primer r!!Template!!KOD neo!!MilliQ!!Total
+
-
|-
+
-
|5||5||2||1||1||1||1||34||50
+
-
|}<br>
+
-
{| class="wikitable"
+
-
!94℃||98℃||68℃||cycles
+
-
|-
+
-
|2min||10sec||10sec||30
+
-
|}<br>
+
-
 
+
-
==September 2==
+
-
 
+
-
====PCR(re;re)====
+
-
 
+
-
first
+
-
{|class="wikitable"
+
-
!buffer!!dNTPs!!MgSO4!!primer f!!primer r!!Template(1ng/µL)!!KODplus neo!!MilliQ!!Total
+
-
|-
+
-
|5||5||3||1||1||1||1||33||50
+
-
|}<br>
+
-
 
+
-
second
+
-
{|class="wikitable"
+
-
!buffer!!dNTPs!!MgSO4!!primer f!!primer r!!Template(10ng/µL)!!KODplus neo!!MilliQ!!Total
+
-
|-
+
-
|5||5||3||1||1||1||1||33||50
+
-
|}<br>
+
-
[[File:IMG_2186.jpg|200px|thumb|right]]
+
-
{| class="wikitable"
+
-
!94℃||98℃||68℃||cycles
+
-
|-
+
-
|2min||10sec||10sec||25
+
-
|}<br>
+
-
 
+
-
Lane1: first Template 1ng<br>
+
-
Lane2: second Template 10ng<br>
+
-
Lane3: 1kb ladder<br>
+
-
refine first Template => 132ng/µL<br>
+
-
 
+
-
==September 3==
+
-
 
+
-
====Restriction enzyme processing====
+
-
 
+
-
{|class="wikitable"
+
-
!FT(132ng/µL)!!bufferM!!EcoRI!!SpeI!!MilliQ!!Total
+
-
|-
+
-
|10||2||1||1||6||20
+
-
|}
+
-
37℃,overnight => refinement 31.6ng/µL (Elution 40µL)<br><br>
+
-
 
+
-
'''Ligation'''<br>
+
-
Insert(FT: 31.6ng/µL, 600bp ):  2µL =  26fmol<br>
+
-
Vector(DT: 28.0ng/µL, 3300bp):  3µL = 240fmol<br>
+
-
Ligation High ver.2          :2.5µL          <br>
+
-
=> 16℃, 2 hours                            <br><br>
+
-
 
+
-
'''Transformation'''<br>
+
-
{|class="wikitable"
+
-
!competent cell!!DNA!!plate!!colony
+
-
|-
+
-
|20µL||FT-DT 2µL||Amp||o
+
-
|-
+
-
|20µL||pT7-6His:R9 2µL||Amp||o
+
-
|-
+
-
|10µL||GFP generator 1µL||Amp||o
+
-
|}(BBa,I746915,pT7-6His:GFP) <br>
+
-
 
+
-
==September 4==
+
-
'''Colony PCR'''<br><br>
+
-
[[File:IMG_2187.jpg|200px|thumb|right]]
+
-
{|class="wikitable"
+
-
!Quick Tag!!VF2!!VR!!MilliQ!!Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
 
+
-
{| class="wikitable"
+
-
!94℃||98℃||55℃||68℃||cycles
+
-
|-
+
-
|2min||30sec||30sec||1min||25
+
-
|}<br>
+
-
Lane1: 1kb ladder<br>
+
-
Lane2: FT-DT(about 700bp)<br>
+
-
 
+
-
Liquid culture(4ml) 23:30 ~<br>
+
-
GFP generator, FT-DT
+
-
 
+
-
==September 5==
+
-
 
+
-
'''Miniprep(FT-DT)'''            <small>by Sato</small><br>
+
-
88.8ng/µL<br><br>
+
-
 
+
-
'''Restriction enzyme processing'''
+
-
{| class="wikitable"
+
-
!FT-DT(88.8ng/µL)||XbaI||PstI||bufferM||MiliiQ||total
+
-
|-
+
-
|20||1||1||4||14||40
+
-
|}
+
-
at 37℃, 2 hours<br>
+
-
 
+
-
'''Electrophoresis'''            <small>by Takeuchi</small><br>
+
-
[[File:IMG_2188.jpg|200px|thumb|right]]
+
-
<br><br><br><br><br><br><br><br><br>
+
-
'''Restriction enzyme processing'''<small>by Takeuchi</small><br>
+
-
 
+
-
[[File:IMG_2189.jpg|200px|thumb|right]]
+
-
{| class="wikitable"
+
-
!FT-DT(88.8ng/µL)||bufferM||XbaI/Spe||MiliiQ||total
+
-
|-
+
-
|4||1||0.5||4.5||10
+
-
|}
+
-
{| class="wikitable"
+
-
!FT-DT||bufferH||Pst/Eco||MiliiQ||total
+
-
|-
+
-
|4||1||0.5||4.5||10
+
-
|}<br>
+
-
at 37℃,1hour 10min
+
-
 
+
-
==September 6==
+
-
'''Western blotting'''(BBa,I746915)<br>
+
-
 
+
-
'''Sample making'''<br>
+
-
SOC(ampt) 50ml + pre culture 1ml    x2              <br>
+
-
OD600 = 0.5~0.7 incubate at 37℃  (OD 0.642)      <br>
+
-
add IPTG final concentration is 1mM (negative control)<br>
+
-
incubate at 37℃,4 hours<br><br>
+
-
 
+
-
'''SDS-PAGE'''<br>
+
-
Do spin down E.coli and make suspension put E.coli into 1mL 1x sample buffer <br>
+
-
95℃,10min<br>
+
-
electrophoresis at 500V, 30mA, 50min<br>
+
-
Lane1: 10µL    (IPTG -)<br>
+
-
Lane2: 10µL    (IPTG -)<br>
+
-
Lane3: 10µL    (IPTG +)<br>
+
-
Lane4:  5µL    (IPTG -)<br>
+
-
Lane5:  5µL    (IPTG +)<br>
+
-
Lane6:  2µL    (IPTG -)<br>
+
-
Lane7:  2µL    (IPTG +)<br><br>
+
-
 
+
-
Blotting at 50V,100mA,30min
+
-
Put into blocking buffer and vibrating 30min<br>
+
-
Incubate with Anti GFP(1/1000) 10mL at RT,1h<br>
+
-
Washing with 10mL TBST (vibrating 10min x2)<br>
+
-
Incubate with Anti-mouseAP(1/1000) 10mL at RT, 30min<br>
+
-
Washing with 10mL TBST (vibrating 10min x3)<br>
+
-
Put NBT,BCIP into dye buffer
+
-
 
+
-
==September 9==
+
-
 
+
-
'''Mutaion of FT'''
+
-
{|class="wikitable"
+
-
!MilliQ!!buffer!!dNTP!!primer f!!primer r!!FT(52ng/µL)!!KODplus!!Total
+
-
|-
+
-
|35||5||5||1.5||1.5||1||1||50
+
-
|}
+
-
{| class="wikitable"
+
-
!94℃||98℃||68℃||cycles
+
-
|-
+
-
|2min||10sec||4min||20
+
-
|}
+
-
→ GeneClean II 32.6ng/µL<br><br>
+
-
 
+
-
'''Dpn1 processing'''
+
-
 
+
-
[[File:IMG_2190.jpg|200px|thumb|right]]
+
-
{|class="wikitable"
+
-
!TA buffer!!DNA!!Dpn1!!Total
+
-
|-
+
-
|3.3||31||2||36.3
+
-
|}<br><br><br><br><br><br><br><br>
+
-
'''Ligation'''
+
-
 
+
-
{|class="wikitable"
+
-
!DNA!!MiliiQ!!Ligation high Ver.2!!T4 kinase!!Total
+
-
|-
+
-
|2||7||5||1||15
+
-
|}
+
-
 
+
-
'''Transformation'''
+
-
competent cell: 10<br>
+
-
DNA          :  1
+
-
 
+
-
==September 10==
+
-
 
+
-
'''Miniprep of FT'''
+
-
①116.9ng/µL<br>
+
-
② 34.5ng/µL<br>
+
-
'''Restriction enzyme processing'''(Mutation checking)
+
-
{|class="wikitable"
+
-
!FT①/②!!bufferHl!!EcoRI!!PstI!!MiliQ!!Total
+
-
|-
+
-
|4||1||0.5||0||4||10
+
-
|-
+
-
|4||1||0||0.5||4||10
+
-
|}
+
-
 
+
-
'''PCR'''
+
-
{|class="wikitable"
+
-
!Bufer!!dNTPs!!MgSO4!!primer(+/-RBS)fwd!!primer(+/-RBS)rev!!template①/②!!KOD plus neo!!MilliQ!!Total
+
-
|-
+
-
|5||5||3||1||1||1||1||33||50
+
-
|}
+
-
{| class="wikitable"
+
-
!94℃||98℃||68℃||cycles
+
-
|-
+
-
|2min||10sec||10sec||25
+
-
|}
+
-
->purifying column 17.4ng/µL<br><br>
+
-
 
+
-
====Restriction enzyme processing====
+
-
{|class="wikitable"
+
-
!FT(RBS+)32.6ng/µL!!Xbal!!Pstl!!buffer M!!BSA!!Total
+
-
|-
+
-
|30||1||1||4||4||40
+
-
|}
+
-
{|class="wikitable"
+
-
!T7-His:R9(62.6ng/µL)!!SpeI!!Pstl!!MilliQ!!buffer M!!Total
+
-
|-
+
-
|15||0.5||0.5||2||2||20
+
-
|}<br>
+
-
incubate at 37℃, 3hours    <br>
+
-
->purifying column 17.4ng/µL <br><br>
+
-
 
+
-
==September 11==
+
-
'''Ligation'''<br>
+
-
Vector(T7: 33.4ng/µL, 2100bp):  1µL =  24fmol<br>
+
-
Insert(FT: 17.4ng/µL, 600bp ):  5µL = 217fmol<br>
+
-
Ligation High ver.2          :  3µL          <br>
+
-
=> 16℃, 1 hours                            <br><br>
+
-
 
+
-
'''Transformation'''<br>
+
-
{|class="wikitable"
+
-
!competent cell!!DNA
+
-
|-
+
-
|20µL||T7-FT 2µL
+
-
|}<br>
+
-
 
+
-
'''PCR(FT without RBS retry)'''
+
-
[[File:IMG_2191.jpg|200px|thumb|right]]
+
-
{|class="wikitable"
+
-
!Bufer!!dNTPs!!MgSO4!!primer(-RBS)fwd!!primer(-RBS)rev!!template!!KOD plus!!MilliQ!!Total
+
-
|-
+
-
|5||5||3||1||1||1||1||33||50
+
-
|}
+
-
 
+
-
{| class="wikitable"
+
-
!94℃||98℃||65℃||68℃||cycles
+
-
|-
+
-
|2min||15sec||30sec||30sec||25
+
-
|}<br>
+
-
Lane1:FT(RBS-) 600bp  <br>
+
-
Lane2:Ladder  100bp  <br>
+
-
=>purifying column 34.6ng/µL<br><br>
+
-
 
+
-
'''Restriction enzyme processing'''
+
-
{| class="wikitable"
+
-
!FT(RBS-)||XbaI||PstI||bufferM||BSA||total
+
-
|-
+
-
|30||1||1||4||4||40
+
-
|}
+
-
at 37℃, 2 hours<br>
+
-
=> 6.2ng/μL<br>
+
-
 
+
-
{| class="wikitable"
+
-
!GFP-DT||XbaI||PstI||bufferM||BSA||MilliQ||total
+
-
|-
+
-
|15||1||1||3||3||7||30
+
-
|}
+
-
at 37℃, 2 hours<br>
+
-
=> 7.0ng/μL<br><br>
+
-
 
+
-
'''Ligation'''<br>
+
-
Vector(T7-6His-R9: 11.6ng/µL, 2100bp):  1µL =  9fmol<br>
+
-
Insert(FT: 6.2ng/µL, 600bp ):  5µL = 79fmol<br>
+
-
Ligation High ver.2          :  3µL          <br>
+
-
=> 16℃, 1 hours                            <br><br>
+
-
 
+
-
Vector(T7-6His-R9: 11.6ng/µL, 2100bp):  1µL =  9fmol<br>
+
-
Insert(GFP-DT: 7.0ng/µL, 1000bp ):  9µL = 95fmol<br>
+
-
Ligation High ver.2          :  4µL          <br>
+
-
=> 16℃, 1 hours                            <br><br>
+
-
 
+
-
==September 12==
+
-
'''Transformation'''<br>
+
-
{|class="wikitable"
+
-
!competent cell!!DNA!!plate
+
-
|-
+
-
|20µL||T7-R9-GFP-DT 2µL||Amp
+
-
|-
+
-
|20µL||T7-R9-FT 2µL||Amp
+
-
|-
+
-
|BL21CDE3 10µL||T7-FT 1µL||Amp
+
-
|}<br><br>
+
-
 
+
-
'''Miniprep (T7-FT)'''<br>
+
-
37.1ng/µL<br><br>
+
-
 
+
-
==September 13==
+
-
'''Miniprep'''<br>
+
-
①T7-R9-GFP-DT 90ng/µL<br>
+
-
②T7-R9-FT    160ng/μL<br><br>
+
-
 
+
-
'''Restriction enzyme processing'''
+
-
{| class="wikitable"
+
-
!T7-R9-GFP-DT (90ng/µL)||EcoRI||PstI||buffer H||MilliQ||total
+
-
|-
+
-
|20||1||1||3||5||30
+
-
|}
+
-
at 37℃, 2 hours<br>
+
-
=> 20.7ng/μL<br>
+
-
{| class="wikitable"
+
-
!T7-R9-FT (160ng/µL)||EcoRI||SpeI||buffer M||MilliQ||total
+
-
|-
+
-
|20||1||1||3||5||30
+
-
|}
+
-
at 37℃, 2 hours<br>
+
-
=> 20.1ng/μL<br><br>
+
-
 
+
-
'''Transformation'''<br>
+
-
{|class="wikitable"
+
-
!competent cell  BL21(DE3)!!DNA!!plate!!
+
-
|-
+
-
|10µL||T7-R9-GFP-DT 1µL||Amp
+
-
|-
+
-
|10µL||T7-R9-FT 1µL||Amp
+
-
|}<br><br>
+
-
 
+
-
'''Restriction enzyme processing'''
+
-
{| class="wikitable"
+
-
!Buffer H||BSA||EcoRI||PstI||DpnⅠ||MilliQ||total
+
-
|-
+
-
|5||5||0.5||0.5||0.5||13.5||25
+
-
|}
+
-
=>We define this solution "2× Master Mix"<br><br>
+
-
 
+
-
{| class="wikitable"
+
-
!2× Master Mix||pSB1C3(Linerarized Plasmid Backbone)
+
-
|-
+
-
|4||4
+
-
|}
+
-
at 37℃, 30 minutes<br>
+
-
80℃, 30 minutes<br><br>
+
-
 
+
-
'''PCR(Insert His-tag)'''
+
-
[[File:IMG_2192.jpg|200px|thumb|right]]
+
-
{|class="wikitable"
+
-
!MilliQ!!Buffer for iPCR!!dNTPs!!primer fwd!!primer rev!!template(T7-FT 37.1ng/μL)!!KOD plus!!Total
+
-
|-
+
-
|35||5||5||1.5||1.5||1||1||50
+
-
|}
+
-
 
+
-
{| class="wikitable"
+
-
!94℃||94℃||56℃||68℃||cycles
+
-
|-
+
-
|2min||15sec||30sec||2.5min||15
+
-
|}<br>
+
-
Lane1:Ladder            1kb  <br>
+
-
Lane2:T7-6His:FT  about 2700bp  <br>
+
-
<br><br>
+
-
 
+
-
==September 14==
+
-
'''Ligation'''<br>
+
-
Vector(pSB1C3: 12.5/µL, 2000bp)        :  2µL =  19fmol<br>
+
-
Insert(T7-R9-GFP-DT: 20.7ng/µL, 700bp ):  4µL = 180fmol<br>
+
-
Ligation High ver.2                    :  3µL          <br>
+
-
=> 16℃, 1 hours      <br><br>
+
-
 
+
-
'''Dpn1 processing'''
+
-
{|class="wikitable"
+
-
!PCR products(9/13)!!Dpn1!!Total
+
-
|-
+
-
|45||2||47
+
-
|}
+
-
=>37℃, 1 hour <br><br>
+
-
 
+
-
'''Self Ligation'''
+
-
{|class="wikitable"
+
-
!PCR products!!Ligation High!!T4 Kinase!!MilliQ!!Total
+
-
|-
+
-
|2||5||1||7||15
+
-
|}
+
-
=>16℃, 1 hour<br><br>
+
-
 
+
-
'''Ligation'''<br>
+
-
Vector(DT: 17.1/µL, 2100bp)        :  2µL =  12fmol<br>
+
-
Insert(T7-R9-FT: 20.1ng/µL, 650bp ):  5µL = 496fmol<br>
+
-
Ligation High ver.2                :  3.5µL          <br>
+
-
=> 16℃, 1 hours      <br><br>
+
-
 
+
-
 
+
-
==September 15==
+
-
'''Restriction enzyme processing'''
+
-
{| class="wikitable"
+
-
!FT without RBS (34.6ng/µL)||EcoRI||PstI||10× buffer H||MilliQ||total
+
-
|-
+
-
|10||0.5||0.5||2||7||20
+
-
|}
+
-
<br><br>
+
-
 
+
-
'''Colony PCR'''<br>
+
-
{|class="wikitable"
+
-
!Quick Tag!!VF!!VR!!MilliQ!!Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
 
+
-
{| class="wikitable"
+
-
!94℃||94℃||55℃||68℃||cycles
+
-
|-
+
-
|2min||30sec||30sec||1min||25
+
-
|}<br><br>
+
-
 
+
-
'''Electrophoresis'''<br>
+
-
Lane1: ladder<br>
+
-
Lane2: T7-R9-GFP-DT<br>
+
-
Lane3: T7-R9-GFP-DT<br><br>
+
-
 
+
-
Lane4: T7-R9-FT-DT<br>
+
-
Lane5: T7-R9-FT-DT<br>
+
-
Lane6: T7-R9-FT-DT<br>
+
-
Lane7: T7-R9-FT-DT<br>
+
-
Lane8: T7-R9-FT-DT<br>
+
-
A member of secretion group electrophoresed DNA from Lane9 to Lane12.<br><br>
+
-
 
+
-
Lane13: T7-His-FT<br>
+
-
Lane14: T7-His-FT<br>
+
-
Lane15: T7-His-FT<br>
+
-
Lane16: T7-His-FT<br>
+
-
Lane17: ladder<br><br>
+
-
 
+
-
<pre style="color:red">えいどうしゃしん</pre>
+
-
<br><br>
+
-
 
+
-
Liquid culture(3ml) 3:30~<br>
+
-
T7-R9-FT-DT×2, T7-His-FT×2, T7-His-FT<br><br>
+
-
 
+
-
==September 16==
+
-
'''Miniprep'''<br>
+
-
①T7-R9-FT-DT  150ng/µL<br>
+
-
②T7-R9-FT-DT  139ng/μL<br>
+
-
③T7-R9-GFP-DT 67ng/µL<br>
+
-
④T7-His-FT    153ng/μL<br>
+
-
⑤T7-His-FT    73ng/μL<br><br>
+
-
'''Electrophoresis'''<br>
+
-
[[File:IMG_2193.jpg|200px|thumb|right]]
+
-
<br><br><br><br><br><br><br><br><br>
+
-
 
+
-
==September 17==
+
-
'''Purifying column'''<br>
+
-
=>FT without RBS :36.4ng/µL<br><br>
+
-
 
+
-
'''Ligation'''<br>
+
-
Vector(PSB1C3: 12.5/µL, 2000bp)          :  3µL =  9fmol<br>
+
-
Insert(FT without RBS: 36.4ng/µL, 600bp ):  3µL = 92fmol<br>
+
-
Ligation High ver.2                      :  3µL <br>
+
-
=> 16℃, 1 hours      <br><br> 
+
-
 
+
-
==September 18==
+
-
'''Transformation'''  <small>by NAKAGAWA</small><br>
+
-
{|class="wikitable"
+
-
!competent cell!!DNA!!plate
+
-
|-
+
-
|20µL||PSB1C3 FT without RBS 1µL||CP+
+
-
|}<br>
+
-
'''Verification of R9 function'''  <small>by TAKEUCHI</small><br>
+
-
{|class="wikitable"
+
-
|R9(20µg/µL)||0.9µL
+
-
|-
+
-
|GFP(1.2mg/mL)||2.23µL
+
-
|-
+
-
|RBS||16.85µL
+
-
|-
+
-
|total||20µL
+
-
|}
+
-
X5<br>
+
-
 
+
-
Method:<br>
+
-
1. Peel cuticles on parafilm by using the head of pencil.(Menasha,wI,54952)<br>
+
-
2. Put plant cells into GFP&R9 or GFP for 5~30min.<br>
+
-
3. Put plant cells into PBS.<br>
+
-
4. Hoechst dyeing.<br>
+
-
 
+
-
{|class="wikitable"
+
-
|||1||2||3||4||5||6
+
-
|-
+
-
|R9||o||o||o||x||o||o
+
-
|-
+
-
|cuticle||o||o||o||o||x||x
+
-
|-
+
-
|soak in GFP||5min||15min||30min||5min||5min||30min
+
-
|}
+
-
 
+
-
==September 19==
+
-
'''Transformation(re)''' <small>by NAKAGAWA,TAKEUCHI</small><br>
+
-
competent cell:20µL<br>
+
-
DNA(No RBS FT):1µL<br>
+
-
plate        :CP+<br><br>
+
-
'''Transformation(re;re)''' <small>by NAKAGAWA,TAKEUCHI</small><br>
+
-
competent cell:10µL<br>
+
-
DNA(No RBS FT):1µL<br>
+
-
LB            :100µL<br>
+
-
plate        :CP+<br><br>
+
-
 
+
-
'''Adjusted GM Agar Medium making''' <small>by TAKEUCHI</small>
+
-
 
+
-
Ingredient of MS medium(SIGMA M5519): 0.88g<br>
+
-
MES                                : 0.1g<br>
+
-
ion exchanged water                : 200mL<br>
+
-
NaOH                                : 26µL(adjust to pH5.6)<br>
+
-
Agar medium                        : 1.6g<br>
+
-
 
+
-
Autoclave 120℃<br>
+
-
 
+
-
====Colony PCR(re)====
+
-
[[File:IMG_2194.jpg|200px|thumb|right]]
+
-
'''gelA'''<br>
+
-
Lane1: 100bp Ladder<br>
+
-
Lane2: No RBS FT colony number 9<br>
+
-
Lane3: No RBS FT colony number 10<br>
+
-
Lane4: No RBS FT colony number 11<br>
+
-
Lane5: No RBS FT colony number 12<br>
+
-
Lane6: No RBS FT colony number 13<br>
+
-
Lane7: empty<br>
+
-
Lane8: empty<br><br><br>
+
-
[[File:IMG_2195.jpg|200px|thumb|right]]<br><br><br><br><br><br><br><br><br><br>
+
-
'''gelB'''<br>
+
-
[[File:IMG_2196.jpg|200px|thumb|right]]
+
-
Lane1: 100bp Ladder<br>
+
-
Lane2: No RBS FT colony number 14<br>
+
-
Lane3: No RBS FT colony number 15<br>
+
-
Lane4: No RBS FT colony number 16<br>
+
-
Lane5: No RBS FT colony number 17<br>
+
-
Lane6: No RBS FT colony number 18<br>
+
-
Lane7: No RBS FT colony number 19<br>
+
-
Lane8: empty<br><br><br><br>
+
-
'''gelC'''<br>
+
-
[[File:IMG_2197.jpg|200px|thumb|right]]
+
-
Lane1: 100bp Ladder<br>
+
-
Lane2: No RBS FT colony number 20<br>
+
-
Lane3: No RBS FT colony number 21<br>
+
-
Lane4: No RBS FT colony number 22<br>
+
-
Lane5: No RBS FT colony number 23<br>
+
-
Lane6: No RBS FT colony number 24<br>
+
-
Lane7: empty<br>
+
-
Lane8: empty<br><br><br><br><br>
+
-
 
+
-
==September 20==
+
-
 
+
-
'''Liquid culture''' <small>by NAKAGAWA</small><br>
+
-
No RBS FT x8 (by using September 18)<br>
+
-
Plate: CP+<br>
+
-
 
+
-
'''Colony PCR'''<br>
+
-
[[File:IMG_2194.jpg|200px|thumb|right]]
+
-
No RBS FT by using September19<br>
+
-
 
+
-
Lane1 : 100bp ladder<br>
+
-
Lane2 : colony number 1<br>
+
-
Lane3 : colony number 2<br>
+
-
Lane4 : colony number 3<br>
+
-
Lane5 : colony number 4<br>
+
-
Lane6 : colony number 5<br>
+
-
Lane7 : colony number 6<br>
+
-
Lane8 : colony number 7<br>
+
-
Lane9 : colony number 8<br>
+
-
Lane10: empty<br>
+
-
Lane11: empty<br>
+
-
Lane12: 100bp ladder<br>
+
-
'''Liquid culture'''<br>
+
-
A-11, A-12<br>
+
-
 
+
-
'''Miniprep'''
+
-
No RBS FT(by using September20)
+
-
1.-10.4µg/mL<br>
+
-
2.-4.7µg/mL<br>
+
-
3.-3.6µg/mL<br>
+
-
4.-7.6µg/mL<br>
+
-
5.-4.5µg/mL<br>
+
-
6.-8.4µg/mL<br>
+
-
7. 1.8µg/mL(average)<br>
+
-
8. 18.1µg/mL<br>
+
-
'''Restriction enzyme processing'''
+
-
{| class="wikitable"
+
-
!DNA(No RBS FT)x2||E.coli||BufferH||MilliQ||total
+
-
|-
+
-
|30µL||1µL||4µL||5µL||40µL
+
-
|}
+
-
in 37℃,2hours<br><br>
+
-
 
+
-
'''Electrophoresis'''<br>
+
-
DNA(No RBS FT) sample7,8: 10µL<br>
+
-
Loading Dye            :  2µL<br><br>
+
-
Lane1: 1kb ladder<br>
+
-
Lane2: empty <br>
+
-
Lane3: No RBS FT(E) sample7<br>
+
-
Lane4: empty<br>
+
-
Lane5: No RBS FT(E) sample8<br>
+
-
Lane6: empty<br>
+
-
[[File:IMG_2198.jpg|200px|thumb|right]]
+
-
'''Electrophoresis(re)'''
+
-
[[File:IMG_2199.jpg|200px|thumb|right]]
+
-
<br>
+
-
 
+
-
'''RNA Extraction'''<br>
+
-
5 leaves: 100mg<br>
+
-
ISOGEN  : 1mL<br>
+
-
Elution : 20µL<br><br>
+
-
 
+
-
'''cDNA Synthesis(1/4)'''<br>
+
-
 
+
-
{|class="wikitable"
+
-
!gDNA wipeout buffer!!template RNA!!H2O!!Total
+
-
|-
+
-
|1µL||0.5µL||5.5µL||7µL
+
-
|}at 42℃, 2min
+
-
 
+
-
{|class="wikitable"
+
-
!Reverse Transcriptase!!RT buffer!!primer mix!!template!!Total
+
-
|-
+
-
|0.5µL||2µL||0.5µL||7µL||10µL
+
-
|}at 42℃, 2min and 95℃,3min<br><br>
+
-
 
+
-
'''RT PCR'''
+
-
{|class="wikitable"
+
-
!buffer!!dNTPs!!MgSO4!!primer f!!primer r!!Template!!KODplus!!MilliQ!!Total
+
-
|-
+
-
|5||5||2||1.5||1.5||1||1||34.5||50
+
-
|}
+
-
{| class="wikitable"
+
-
!94℃||94℃||54℃||68℃||cycles
+
-
|-
+
-
|2min||15sec||30sec||10sec||30
+
-
|}<br>
+
-
1.TUBULIN<br>
+
-
2.FUL<br>
+
-
3.SEP3<br>
+
-
4.AP1<br>
+
-
[[File:IMG_2200.jpg|200px|thumb|right]]<br><br><br><br><br><br><br><br><br><br><br><br>
+
-
==September 22==
+
-
'''mRNA extraction'''<br>
+
-
FT and R9<br>
+
-
Arabidopsis thaliana's leaves : 200mg
+
-
{|class="wikitable"
+
-
!R9||FT||total
+
-
|-
+
-
|1.575μL||33.425μL||35μL
+
-
|}
+
-
{|class="wikitable"
+
-
!R9||GFP
+
-
|-
+
-
|1.575||33.425||35
+
-
|}
+
-
1. Strip cuticles on palafilm by the head of a pencil.<br>
+
-
2. Soak plant cells in R9 & FT or R9 & GFP for 2 hours(19:45~).<br>
+
-
3. Add PBS (21:45~)<br><br>
+
-
'''Microscope'''<br>
+
-
R9+GFP+Plant Cell ver.3<br>
+
-
R9+
+
-
{|class="wikitable"
+
-
!R9||GFP||PBS||total
+
-
|-
+
-
|0.9||2.25||16.85||20μL
+
-
|}
+
-
R9-
+
-
{|class="wikitable"
+
-
!R9||GFP||PBS||total
+
-
|-
+
-
|0||2.25||17.65||20
+
-
|}
+
-
same as 9/20, only not on parafilm but on microscope slide from the start.
+
-
 
+
-
==September 23==
+
-
[[File:data0923.jpg|200px|thumb|right]]
+
-
'''RNA Extraction'''<br>
+
-
FT- (GFP+)
+
-
FT+
+
-
{|class="wikitable"
+
-
!ISOGEN||chloroform||elution||total
+
-
|-
+
-
|1||200||11||212
+
-
|}
+
-
'''Reverce Transcription'''<br>
+
-
{|class="wikitable"
+
-
|gDNA wipeout buffer||RNA||total
+
-
|-
+
-
|1||6||7
+
-
|}
+
-
at 42 degree, for 2min.
+
-
{|class="wikitable"
+
-
!Reverce Transcriptase||RT buffer||primer mix||Template||total
+
-
|-
+
-
|0.5||2||0.5||7||10
+
-
|}
+
-
at 42 degree, for 30min.<br>
+
-
at 95 degree, for 3min.<br><br>
+
-
'''RT-PCR'''
+
-
{|class="wikitable"
+
-
!Buffer||dNTPs||MgSO4||primer||Template||KOD Plus||MilliQ||total
+
-
|-
+
-
|5||5||2||1.5||1||1||34.5
+
-
|}
+
-
94℃, 2min<br>
+
-
94℃, 15sec<br>
+
-
54℃, 30sec<br>
+
-
68℃, 10sec<br><br>
+
-
1.NC TUBULIN<br>
+
-
2.  FUL<br>
+
-
3.  SEP3<br>
+
-
4.  AP1<br>
+
-
5.FT TUBULIN<br>
+
-
6.  FUL<br>
+
-
7.  SEP3<br>
+
-
8.  AP1<br><br>
+
-
 
+
-
==September 24==
+
-
'''ScreeningPCR & Electrophoresis'''<br>
+
-
[[File:ElectrophoresisFFE0924.jpg|200px|thumb|right]]
+
-
Lane<br>
+
-
1. 100bp ladder<br>
+
-
2. T7-His-FT ① 9.2ng/μL<br>
+
-
3. T7-His-FT ② 11.7ng/μL<br>
+
-
4. T7-His-FT ③ 23.6ng/μL<br>
+
-
5. T7-His-FT ④ 21.6ng/μL<br>
+
-
6. T7-His-FT ⑤ 30.6ng/μL<br>
+
-
7. T7-His-FT ⑥ 51.5ng/μL<br>
+
-
8. T7-His-FT ⑦ 47.1ng/μL<br>
+
-
9. T7-His-FT(A4) 20.0ng/μL<br>
+
-
10. T7-His-FT(B1) 18.8ng/μL<br>
+
-
11. final2(by_Secretion_group)<br>
+
-
12. blank<br><br>
+
-
'''FT Introduce'''<br>
+
-
{|class="wikitable"
+
-
!FT||R9||PBS||total
+
-
|-
+
-
|50||2.88||11.12||64
+
-
|}
+
-
{|class="wikitable"
+
-
!GFP||R9||PBS||total
+
-
|-
+
-
|7.2||2.88||53.02||64
+
-
|}
+
-
We took 1 leaf out of 6 individuals of Arabidopsis thaliana that grow three weeks one by one<br>
+
-
Leaves 30mg<br>
+
-
1.cut off leaves<br>
+
-
2.Inject the juice(by terumo-syringe)(Center tip、tuberculin 1nl)<br>
+
-
3.store 20 min, then add PBS 600μL<br>
+
</div>
</div>
-
 
+
<div id="kyoto-tab-NoteGGA">
-
<html><a id="SecretionNotebook"></a></html>
+
{{Kyoto/Notebook/GoldenGate}}
-
 
+
-
=[[File:Kyoto_SecretionNotebook.png|link=]]=
+
-
<div class="_kyoto-note">
+
-
==February 7==
+
-
====Preculture====
+
-
We started preculture at 12:10.<br>
+
-
 
+
-
==February 8==
+
-
====Liquid culture====
+
-
We start culturing with 300mL of LB medium.<br>
+
-
{|class="wikitable"
+
-
!time||OD600
+
-
|-  
+
-
|12:00||start
+
-
|-
+
-
|14:10||0.019
+
-
|-
+
-
|14:45||0.154
+
-
|-
+
-
|15:05||0.267
+
-
|-
+
-
|15:21||0.64
+
-
|}
+
-
 
+
-
====Making Competent Cell====
+
-
We made competent cells.<br><br>
+
-
====Transformation====
+
-
pGEM_TAP<br>LacP (BBa_R0011)<br>DT (BBa_B0015)<br><br>
+
-
====Making Culture Medium Plates====
+
-
We made 200mL of ampicillin culture, kanamycin culture, and chloramphenicol culture.<br><br>
+
-
====Transformation====
+
-
GFP(BBa_E0040) in pSB1A2<br>DT(BBa_B0015) in pSB1AK3<br>ara(BBa_I0500) in pSB2K3<br>LacP(BBa_R0011) in pSB1A2
+
-
 
+
-
==February 9==
+
-
====Transformation====
+
-
BBa-E0040(GFP)(Mr.Fujita)<br><br>
+
-
 
+
-
====Liquid culture====
+
-
DT, LacP colony transformed on February 8<br>
+
-
colony of competent cell made on February 8<br>
+
-
B0040 1.4k pSB1A2 B0034 1.2M pSB1A2(from iGEM parts plate)<br><br>
+
-
 
+
-
====Making Competent cells====
+
-
We did preculture for overnight. We put 1.5mL of preculture on 150mL of LB culture.
+
-
{|class="wikitable"
+
-
!time||OD600
+
-
|-
+
-
|11:45||start
+
-
|-
+
-
|13:30||0.048
+
-
|-
+
-
|14:30||0.168
+
-
|-
+
-
|15:03||0.256
+
-
|-
+
-
|15:20||0.405
+
-
|-
+
-
|15:35||0.459
+
-
|-
+
-
|at last||0.576
+
-
|}
+
-
 
+
-
==February 11==
+
-
====Checking Transformation efficiency====
+
-
Conpetent cell's transformation efficiency is 1.3x10^4colonys/μg<br><br>
+
-
 
+
-
==February 13==
+
-
====Transformation====
+
-
Const promoter J23110, J23109, J23100<br>  
+
-
{|class="wikitable"
+
-
!DNA||Competent cell||Total
+
-
|-
+
-
|1μL||20||21
+
-
|}
+
-
No colony was there on February 14<br><br>
+
-
====Liquid culture====
+
-
LacP, DT, RBS(BBa_B0034),GFP<br>
+
-
start at 20:00<br>
+
-
in Plus grow with Ampicilin 3mL<br>
+
-
 
+
-
==February 14==
+
-
====Miniprep====
+
-
{|class="wikitable"
+
-
|DNA||concentration[μg/μL]
+
-
|-
+
-
|LacP3||39.6
+
-
|-
+
-
|LacP4||40.8
+
-
|-
+
-
|LacP5||28.9
+
-
|-
+
-
|RBS1||28.2
+
-
|-
+
-
|RBS2||57.4
+
-
|-
+
-
|RBS3||13.2
+
-
|-
+
-
|DT3||69.7
+
-
|-
+
-
|DT4||64.4
+
-
|-
+
-
|DT5||61.5
+
-
|-
+
-
|GFP1||64.0
+
-
|-
+
-
|GFP2||50.5
+
-
|-
+
-
|GFP3||66.0
+
-
|}
+
-
 
+
-
====Restrictive Digestion====
+
-
Const promoter J23100<br>
+
-
{|class="wikitable"
+
-
!DNA||Spe1||Pst1||Buffer2||BSA||MilliQ||Total
+
-
|-
+
-
|20||0.5||0.5||3||0.5||5.5||30
+
-
|}
+
-
at 37℃ for overnight<br>
+
-
 
+
-
==February 15==
+
-
====Making gel====
+
-
1% Agarose gel<br>
+
-
{|class="wikitable"
+
-
!Agarose||TAE
+
-
|-
+
-
|1.6g||160mL
+
-
|}
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis021501.JPG|200px|thumb|right|No.1]]
+
-
Gel No.1
+
-
{|class="wikitable"
+
-
!Restriction product||loading dye
+
-
|-
+
-
|5μL||1
+
-
|}
+
-
The marker was 1kb ladder<br>
+
-
It seemed that this restriction product was not cut.<br><br><br><br><br><br>
+
-
[[File:Electrophoresis021502.JPG|200px|thumb|right|No.2]]
+
-
Gel No.2<br>
+
-
Lane1 : 1kb ladder<br>
+
-
Lane2 : J23100 2μL + 6*Loading dye 1μL<br>
+
-
Lane3 : J23100(Spe1,Pst1) 5μL<br>
+
-
Lane4 : J23100(Spe1,Pst1) 2μL<br>
+
-
*There were bands on lane_2 and we cannot identify these bands because the sample of lane_2 was not cut with any restriction enzyme.<br>
+
-
*There must have been bands at 2100bp and 883bp on lane_3 and lane_4.<br><br>
+
-
 
+
-
====Testing whether restriction enzyme were deactivated or not====
+
-
{|class="wikitable"
+
-
!DNA(DT)||restriction enzyme||Buffer||BSA||MilliQ||Total
+
-
|-
+
-
|10||0.5||3||0.5||16||30
+
-
|}
+
-
at 37℃ for Oveernight<br>
+
-
Restriction enzyme means Spe1(1, 2) Pst1(1, 2, 3) in this time.<br>
+
-
 
+
-
==February 16==
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis021601.JPG|200px|thumb|right]]
+
-
1. 1kb ladder<br>
+
-
2. DT2<br>
+
-
3. DT3<br>
+
-
4. DT2 (Spe1-1)<br>
+
-
5. DT2 (Spe1-2)<br>
+
-
6. DT2 (Pst1-1)<br>
+
-
7. DT2 (Pst1-2)<br>
+
-
8. DT2 (Pst1-3)<br>
+
-
9. DT3 (Pst1-4)<br>
+
-
10. 1kb ladder<br><br>
+
-
Pst1-1, Pst1-2, and Pst1-3 did not cut DNAs. They seemed to be deactivated.<br>
+
-
 
+
-
====Genomic PCR====
+
-
{|class="wikitable"
+
-
!10*Buffer for KOD Plus||2mM dNTPs||25mM MgSO4||10μM primer-f||10μM primer-r||158ng/μL Genomic DNA||KOD plus||MilliQ||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||1||32||50
+
-
|}  
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis021602.JPG|200px|thumb|right]]
+
-
1. 1kb ladder<br>
+
-
2. tatABCD (2.5kb)<br>
+
-
3. TAMO reductase (2.7kb)<br>
+
-
4. Negative control<br>
+
-
We got bands of tatABCD but there were nonspecific amplification products.<br>
+
-
We failed amplification of TAMO reductase.<br><br><br><br><br><br>
+
-
 
+
-
====Transformation====
+
-
Constitutive promoter (BBa_J23107, BBA_J23117)<br>
+
-
High copy plasmid (pSB1AT3)<br>
+
-
{|class="wikitable"
+
-
!DNA||competent cell
+
-
|-
+
-
|1μL||10μL
+
-
|}
+
-
 
+
-
==February 17==
+
-
====PCR====
+
-
We did PCR to amplify products of PCR that we had done yesterday but we could not amplify tatABCD.<br><br>
+
-
====Genomic PCR====
+
-
{|class="wikitable"
+
-
!Buffer||dNTPs||MgSO4||primer-f||primer-r||genomic DNA||KOD plus||MilliQ||Total
+
-
|-
+
-
|2.5||2.5||5||0.75||0.75||0.5||0.5||16||50
+
-
|}
+
-
Predenature  94℃,  2min<br>
+
-
Denature      98℃,  10sec<br>
+
-
Annealing    57℃,  30sec<br>
+
-
Extension    68℃, 2.5min<br>
+
-
(30cycles)<br><br>
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis021701.JPG|200px|thumb|right]]
+
-
1. 1kb ladder<br>
+
-
2. TAMO reductase<br>
+
-
3. Negative control<br><br><br><br><br><br>
+
-
 
+
-
====Restrictive Digestion====
+
-
{|class="wikitable"
+
-
!J23100||Spe1||Pst1||Buffer2||BSA||MilliQ||Total
+
-
|-
+
-
|10||0.5||0.5||3||0.5||15.5||30
+
-
|}
+
-
====Genomic PCR====
+
-
{|class="wikitable"
+
-
! ||Buffer||dNTPs||MgSO4||primer-f||primer-r||genomic DNA||KOD plus||MilliQ||Total
+
-
|-
+
-
|TMAO reductase||2.5||2.5||3||0.75||0.75||0.5||0.5||15.5||25
+
-
|-
+
-
|tatABCD||2.5||2.5||2||0.75||0.75||0.5||0.5||15.5||25
+
-
|-
+
-
|tatABCD||2.5||2.5||2||0.75||0.75||5||0.5||10.5||25
+
-
|}
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis021702.JPG|200px|thumb|right]]
+
-
1. 1kb ladder <br>
+
-
2.3.  TAMO reductase<br>
+
-
4. tatABCD<br>
+
-
5. tatABCD(10 times amount of genome)<br><br><br><br>
+
-
 
+
-
====Checking of restriction enzyme====
+
-
{|class="wikitable"
+
-
!DT||enzyme||Buffer||BSA||MilliQ||Total
+
-
|-
+
-
|2||0.5||3||0.5||24||30
+
-
|}
+
-
at 37℃ for overnight<br>
+
-
We checked EcoR1 and Xba1.<br>
+
-
 
+
-
==February 18==
+
-
====Miniprep====
+
-
{|class="wikitable"
+
-
! ||μg/mL||260/280||230/260
+
-
|-
+
-
|JS3117-1||135||1.5||2.06
+
-
|-
+
-
|JS3117-2||75||1.6||1.63
+
-
|-
+
-
|JS3109-1||115||1.5||1.88
+
-
|-
+
-
|JS3109-2||75||1.65||1.71
+
-
|-
+
-
|pSB1AT3-1||70||1.66||1.83
+
-
|-
+
-
|pSB1AT3-2||100||1.52||1.54
+
-
|}
+
-
diluted to 25 times<br><br>
+
-
====Making Competent cells====
+
-
We put 3mL of preculture product on yesterday onto 300mL of LB medium
+
-
{|class="wikitable"
+
-
!time||OD600
+
-
|-
+
-
|10:30||start
+
-
|-
+
-
|12:10||0.118
+
-
|-
+
-
|13:00||0.270
+
-
|-
+
-
|13:30||0.502
+
-
|}
+
-
====Transformation====
+
-
{|class="wikitable"
+
-
!pSB1AT3-2||competent cell||MilliQ||Total
+
-
|-
+
-
|0||20||10||30
+
-
|-
+
-
|2||20||8||30
+
-
|-
+
-
|10||20||0||30
+
-
|}
+
-
*Results(on Feb. 19)<br>
+
-
{|class="wikitable"
+
-
!pSB1AT3-2||number of colony
+
-
|-
+
-
|0||0
+
-
|-
+
-
|2||177
+
-
|-
+
-
|10||590
+
-
|}
+
-
Transformation efficiency      7.4x10^4 colonys/μg<br>
+
-
 
+
-
==February 20==
+
-
====Restrictive Digestion====
+
-
sample 1<br>
+
-
{|class="wikitable"
+
-
!DT plasmid||EcoR1||Xba1||Buffer||BSA||MilliQ||Total
+
-
|-
+
-
|7.5||0.5||0.5||3||0.5||18||30
+
-
|}
+
-
sample2<br>
+
-
{|class="wikitable"
+
-
!GFP plasmid||EcoR1||Spe1||Buffer||BSA||MilliQ||Total
+
-
|-
+
-
|10||0.5||0.5||3||0.5||15.5||30
+
-
|}
+
-
====Electrophoresis====
+
-
*sample1<br>
+
-
[[File:Electrophoresis022001_.JPG|200px|thumb|right]]
+
-
a. sample1 2μL + MilliQ 3μL + 6×Loading Dye 1μL<br>
+
-
b. sample1 5μL + 6×Loading Dye 1μL<br>
+
-
lane1. 1kb ladder<br>
+
-
lane2. a<br>
+
-
lane3. b<br>
+
-
lane4. a<br>
+
-
lane5. b<br>
+
-
lane6. a<br>
+
-
lane7. b<br>
+
-
lane8. 1kb ladder<br>
+
-
*sample2<br>
+
-
[[File:Electrophoresis022302.JPG|200px|thumb|right]]
+
-
c. sample2 2μL + MilliQ 3μL + 6×Loading Dye 1μL<br>
+
-
d. sample2 5μL + 6×Loading Dye 1μL<br>
+
-
lane1. 1kb ladder<br>
+
-
lane2. c<br>
+
-
lane3. d<br>
+
-
lane4. 1kb ladder<br><br>
+
-
 
+
-
====PCR====
+
-
{|class="wikitable"
+
-
! ||Buffer||dNTPs||MgSO4||primer-f||primer-r||genomic DNA||KOD plus||MilliQ||Total
+
-
|-
+
-
|tatABCD1||2.5||2.5||1.5||0.75||0.75||0.5||0.5||16||25
+
-
|-
+
-
|tatABCD2||2.5||2.5||1.5||0.75||0.75||0.5||0.5||16||25
+
-
|-
+
-
|TMAO reductase1||2.5||2.5||2||0.75||0.75||0.5||0.5||15.5||25
+
-
|-
+
-
|TMAO reductase2||2.5||2.5||2||0.75||0.75||5||0.5||10.5||25
+
-
|}
+
-
Predenature 94℃    2min<br>
+
-
Denature 98℃    10sec<br>
+
-
Annealing 59℃    30sec<br>
+
-
Extension 68℃    2.5min<br>
+
-
→30cycles<br><br>
+
-
{|class="wikitable"
+
-
! ||Buffer||dNTPs||MgSO4||primer-f||primer-r||PCR products||genomic DNA||KOD plus||MilliQ||Total
+
-
|-
+
-
|tatABCD1||2.5||2.5||1.5||0.75||0.75||0||0.5||0.5||16||25
+
-
|-
+
-
|tatABCD2||2.5||2.5||1.5||0.75||0.75||1||0||0.5||15.5||25
+
-
|-
+
-
|TMAO reductase1||2.5||2.5||2||0.75||0.75||0||0||0.5||16||25
+
-
|-
+
-
|TMAO reductase2||2.5||2.5||2||0.75||0.75||0||0||0.5||16||25
+
-
|}
+
-
Predenature 94℃    2min<br>
+
-
Denature 98℃    10sec<br>
+
-
Annealing 59℃    30sec<br>
+
-
Extension 68℃    2.5min<br>
+
-
→35cycles<br><br>
+
-
 
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis022003.JPG|200px|thumb|right]]
+
-
[[File:Electrophoresis022004.JPG|200px|thumb|right]]
+
-
lane1.1kb ladder<br>
+
-
lane2.tatABCD<br>
+
-
lane3.tatABCD<br>
+
-
lane4.TAMO1<br>
+
-
lane5.TAMO2<br>
+
-
lane6.1kb ladder<br><br><br><br><br>
+
-
lane1.1kb ladder<br>
+
-
lane2.tatABCD1<br>
+
-
lane3.tatABCD2<br>
+
-
lane4.TAMO<br>
+
-
lane5.TAMO<br>
+
-
lane6.1kb ladder<br><br>
+
-
 
+
-
==February 21==
+
-
====PCR (Advantage HF protocol)====
+
-
{|class="wikitable"
+
-
! ||buffer||dNTPs||primer-f||primer-r||gDNA||PCR products||DW||polymerase||Total
+
-
|-
+
-
|tatABCD||2.5||2.5||0.75||0.75||1||0||17||0.5||25
+
-
|-
+
-
|TMAO||2.5||2.5||0.75||0.75||0||1||17||0.5||25
+
-
|}
+
-
Predenature  94℃    1min<br>
+
-
Denature    94℃    30sec<br>
+
-
Annealing    58℃    30sec<br>
+
-
Extension    68℃    3min<br>
+
-
→25cycles<br><br>
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis0221.JPG|200px|thumb|right]]
+
-
1. 1kb ladder  2μL<br>
+
-
2. tatABCD  5μL + 6×Loading Buffer  1μL<br>
+
-
3. TAMO  5μL + 6×Loading Buffer  1μL<br>
+
-
4. 1kb ladder  2μL<br><br>
+
-
====Liquid culture====
+
-
pSB3C5-1,2<br>
+
-
pSB4K5-1,2
+
-
 
+
-
==February 22==
+
-
====Gel extraction====
+
-
lane 1 of the gel 45.0μg/mL<br><br>
+
-
====PCR purification====
+
-
product  38.2μg/mL <br><br>
+
-
====PCR====
+
-
TMAO reductase<br>
+
-
{|class="wikitable"
+
-
! ||Buffer||dNTPs||MgSO4||prefix primer-f||suffix primer-r||product of gel extract||product of PCR purification(1ng/μL)||KOD plus||MilliQ||Total
+
-
|-
+
-
|1||5||5||4||1.5||1.5||0.5||0||1||32.5||50
+
-
|-
+
-
|2||5||5||4||1.5||1.5||1||0||1||32.5||50
+
-
|-
+
-
|3||5||5||4||1.5||1.5||0||0.5||1||32.5||50
+
-
|-
+
-
|4||5||5||4||1.5||1.5||0||1||1||32.5||50
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
59℃, 30sec<br>
+
-
68℃, 3min<br>
+
-
→25cycles<br><br>
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis022201.JPG|200px|thumb|right]]
+
-
1. 1kb ladder<br>
+
-
2. TAMO1<br>
+
-
3. TAMO2<br>
+
-
4. TAMO3<br>
+
-
5. TAMO4<br>
+
-
6. constructive promoter 1-18C<br>
+
-
7. constructive promoter Spe1<br>
+
-
8. constructive promoter Pst1<br>
+
-
9. 1kb ladder<br>
+
-
====PCR====
+
-
{|class="wikitable"
+
-
! ||Buffer||dNTPs||MgSO4||prefix primer-f||suffix primer-r||product of PCR purification(1ng/μL)||KOD plus||MilliQ||Total
+
-
|-
+
-
|1||5||5||3||1.5||1.5||0.5||1||32.5||50
+
-
|-
+
-
|2||5||5||3||1.5||1.5||1||1||32||50
+
-
|-
+
-
|3||5||5||3||1.5||1.5||2||1||31||50
+
-
|-
+
-
|4||5||5||3||1.5||1.5||3||1||30||50
+
-
|-
+
-
|5||5||5||3||1.5||1.5||10||1||29||50
+
-
|-
+
-
|6||5||5||3||1.5||1.5||0||1||33||50
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
59℃, 30sec<br>
+
-
68℃, 3min<br>
+
-
→25cycles<br><br>
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis022202.JPG|200px|thumb|right]]<br><br><br><br><br><br><br><br>
+
-
 
+
-
====Checking Dpn1====
+
-
{|class="wikitable"
+
-
!Buffer||LacP(28.7ng/μL)||Dpn1||MilliQ||Total
+
-
|-
+
-
|2||10||0.5||7.5||20
+
-
|-
+
-
|2||10||0||5||17
+
-
|}
+
-
 
+
-
==February 23==
+
-
====Colony PCR====
+
-
tatABCD(2 samples)<br>
+
-
{|class="wikitable"
+
-
!Buffer||dNTPs||MgSO4||primer-f||primer-r||KOD plus||MilliQ||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||33||50
+
-
|}
+
-
Predenature 94℃  1min<br>
+
-
Denature    98℃ 10sec<br>
+
-
Annealing  59℃ 30sec<br>
+
-
Extension  68℃  3min<br>
+
-
→25cycles<br><br>
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis022301.JPG|200px|thumb|right]]
+
-
1. 1kb ladder 2μL<br>
+
-
2. tatABCD 1  5μL + 6×Loading Buffer  1μL<br>
+
-
3. tatABCD 2  5μL + 6×Loading Buffer  1μL<br>
+
-
4. 1kb ladder 2μL<br><br>
+
-
====Miniprep====
+
-
pSB4K5 and pSB3C5<br>
+
-
deluted it to 25 times and then measured it<br>
+
-
pSB4K5 1 : 60.0 μg/ml      1.67(260/280)      1.98(260/230) <br>
+
-
pSB4K5 2 : 55.0 μg/ml      1.49(260/280)      1.62(260/230) <br>
+
-
pSB3C5-3 : 3.6 μg/ml        1.57(260/280)      3.00(260/230) <br>
+
-
pSB3C5-4 : 1.3 μg/ml        1.44(260/280)      1.04(260/230) <br><br>
+
-
====Liquid culture====
+
-
pSB3C5-3,4<br><br>
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis022302.JPG|200px|thumb|right]]
+
-
1. 1kb ladder 2μL<br>
+
-
2. pSB3C5-3 5μL, 6×loading dye 1μL<br>
+
-
3. pSB3C5-4 5μL, 6×loading dye 1μL<br>
+
-
4. 1kb ladder 2μL<br><br><br>
+
-
 
+
-
==February 27==
+
-
====Test of Dpn1====
+
-
{| class="wikitable"
+
-
!Buffer2||GFP2||BSA||MilliQ||Dpn1
+
-
|-
+
-
|3||3||0.3||23||1
+
-
|}
+
-
====Colony PCR====
+
-
{| class="wikitable"
+
-
! ||buffer||dNTPs||MgSO4||Primer-f||Primer-r||MilliQ||KOD Plus||Total
+
-
|-
+
-
|Colony PCR(2 samples)||5||5||3||1.5||1.5||33||1||50
+
-
|-
+
-
|Negative control||5||5||3||1.5||1.5||34||0||50
+
-
|}
+
-
Predenature 94℃ 2min<br>
+
-
Denature 98℃ 10sec<br>
+
-
Annealing 59℃ 30sec<br>
+
-
Extension 68℃ 3min<br>
+
-
→25cycles<br><br>
+
-
 
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis0227.JPG|200px|thumb|right]]
+
-
{| class="wikitable"
+
-
!lane||DNA||sample||Loading Dye||MilliQ
+
-
|-
+
-
|1||1kb ladder||2||0||0
+
-
|-
+
-
|2||product of PCR1||5||1||0
+
-
|-
+
-
|3||product of PCR2||5||1||0
+
-
|-
+
-
|4||product of PCR(Negative control)||5||1||0
+
-
|-
+
-
|5||product of PCR(2/23)||5||1||0
+
-
|-
+
-
|6||GFP2(DPN1)||10||2||0
+
-
|-
+
-
|7||GFP2||3||2||7
+
-
|-
+
-
|8||1kb ladder||2||0||0
+
-
|}
+
-
 
+
-
====Results of liquid culture====
+
-
We measure this after dilute it to 10 times.<br>
+
-
{|class="wikitable"
+
-
!pSB3C5-5||pSB3C5-6||pSB3C5-5(1% glucose)||pSB3C5-6(1% glucose)
+
-
|-
+
-
|8.5[µg/ml]||-1.8||-17.9||-18.2
+
-
|}
+
-
====PCR====
+
-
{| class="wikitable"
+
-
! ||buffer||dNTPs||MgSO4||Primer-f(prefix)||Primer-r(suffix)||PCR purification product(1ng/µL)||MilliQ||KOD Plus||Total
+
-
|-
+
-
|1||5||5||3||1.5||1.5||0.2||32.8||1||50
+
-
|-
+
-
|2||5||5||3||1.5||1.5||0.5||32.5||1||50
+
-
|}
+
-
*PCR purification product was that purification product(75ng/µL) of electrophoresis-3 deluted to 1ng/µL<br>
+
-
Predenature 94℃,2min<br>
+
-
Denature 98℃,10sec<br>
+
-
Annealing 59℃,30sec<br>
+
-
Extension 68℃,3min<br>
+
-
→25cycles<br><br>
+
-
 
+
-
==February 28==
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis022801.JPG|200px|thumb|right]]
+
-
1. 1kb ladder<br>
+
-
2. PCR1 →Product of gel extraction : tatABCD with prefix and suffix 105[ng/µL]<br>
+
-
3. PCR2<br><br>
+
-
 
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!Buffer2||plasmid(?)||enzyme||MilliQ||Total
+
-
|-
+
-
|2||2||0.2||15.8||20
+
-
|}
+
-
incubate 1 hour at 37℃<br><br>
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis022802.JPG|200px|thumb|right]]
+
-
[[File:Electrophoresis022803.JPG|200px|thumb|right]]
+
-
1. 1kb ladder<br>
+
-
2. Control (without enzymes)<br>
+
-
3. EcoR1<br>
+
-
4. Xba1 (crystallized)<br>
+
-
5. Xba1 (with seal)<br>
+
-
6. Spe1<br>
+
-
7. Pst1<br>
+
-
8. 1kb ladder<br><br>
+
-
====PCR and Electrophoresis====
+
-
{| class="wikitable"
+
-
!Quick Taq Dye Mix||primer-f||primer-r||template||MilliQ||Total
+
-
|-
+
-
|25||1.0||1.0||0.5||22.5||50
+
-
|}
+
-
Predenature 94℃,2min<br>
+
-
Denature 94℃,30sec<br>
+
-
Annealing 59℃,30sec<br>
+
-
Extension 68℃,3min<br>
+
-
→25cycles<br>
+
-
 
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!BufferH||tatABCD||EcoR1||Spe1||MilliQ||Total
+
-
|-
+
-
|2||5||0.2||0.2||12.6||20
+
-
|}
+
-
====PCR purification====
+
-
We eluted the product for 30µL MilliQ<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!Insert(tatABCD)||Vector(pSB1C3)||Ligation High||Total
+
-
|-
+
-
|10||1||5||16
+
-
|}
+
-
4℃, overnight
+
-
 
+
-
==February 29==
+
-
====Transformation====
+
-
{|class="wikitable"
+
-
!tatABCD||competent cell||Total
+
-
|-
+
-
|1||10||11
+
-
|}
+
-
====Checking Restriction enzyme====
+
-
{|class="wikitable"
+
-
!plasmid seems to be 1-18C promoter||Enzyme||Buffer||MilliQ||Total
+
-
|-
+
-
|2||0.2||2||15.8||20
+
-
|}
+
-
====Checking tatABCD====
+
-
{| class="wikitable"
+
-
!tatABCD||Hind3||Buffer||MilliQ||Total
+
-
|-
+
-
|5||0.2||2||12.8||20
+
-
|}
+
-
====PCR====
+
-
{| class="wikitable"
+
-
! ||buffer||dNTPs||MgSO4||Primer-f||Primer-r||ColE1(6.5ng/µL) / TMAO||MilliQ||KOD Plus Neo||Total
+
-
|-
+
-
|Kil||2.5||2.5||1.5||0.75||0.75||0.5||16||0.5||25
+
-
|-
+
-
|TMAO||2.5||2.5||1.5||0.75||0.75||0.5||16||0.5||25
+
-
|}
+
-
Predenature 94℃,2min<br>
+
-
Denature 98℃,10sec<br>
+
-
Annealing 60℃,30sec<br>
+
-
Extension 68℃,3min<br>
+
-
→30cycles<br><br>
+
-
====Electrophoresis====
+
-
[[File:38f7cf5b-20b9-42d0-a497-1885f1f92bbe.jpg|200px|thumb|right]]
+
-
1. 1kb ladder<br>
+
-
2. Kil (649bp)<br>
+
-
3. TMAO (2720bp)<br>
+
-
4. TMAO (Quick Taq)<br>
+
-
5. tatABCD (Quick Taq)<br>
+
-
6. tatABCD (Hind3)<br>
+
-
7. 1kb ladder<br><br>
+
-
 
+
-
==March 1==
+
-
====PCR====
+
-
*TMAO<br>
+
-
Template is gDNA and product of colony PCR gel extraction<br>
+
-
{| class="wikitable"
+
-
! ||Buffer||gNTPs||MgSO4||Primer-f||Primer-r||KOD Plus Neo||Template gDNA||product of gel extraction||DW||Total
+
-
|-
+
-
|1||2.5||2.5||1.5||0.75||0.75||0.5||0.5||0||16||25
+
-
|-
+
-
|2||2.5||2.5||1.5||0.75||0.75||0.5||0||2||14.5||25
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
60℃, 30sec<br>
+
-
68℃, 1.5min<br>
+
-
→25cycles<br><br>
+
-
*Kil
+
-
{| class="wikitable"
+
-
!Buffer||dNTPs||MgSO4||primer-f||primer-r||colE1(6.5ng/µL)||KOD Plus Neo||MilliQ||Total
+
-
|-
+
-
|2.5||2.5||1.5||0.75||0.75||0.5||16||0.5||25
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
61℃, 30sec<br>
+
-
68℃, 1min<br>
+
-
→20cycles<br><br>
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis030101.JPG|200px|thumb|right]]
+
-
1. 1kb ladder<br>
+
-
2. TMAO1 (gDNA)<br>
+
-
3. TMAO2 (product of gel extraction)<br>
+
-
4. Kil<br><br><br><br><br>
+
-
 
+
-
====PCR====
+
-
*kil
+
-
{| class="wikitable"
+
-
!Buffer||dNTPs||MgSO4||primer-f||primer-r||Product of Purification||KOD Plus Neo||MilliQ||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||32||1||50
+
-
|}
+
-
[[File:Electrophoresis030102.JPG|200px|thumb|right]] 
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
61℃, 30sec<br>
+
-
68℃, 30sec<br>
+
-
20cycles<br>
+
-
→Purification 230ng/µL<br><br><br>
+
-
 
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!Kil||EcoR1||Spe1||BufferH||MilliQ||Total
+
-
|-
+
-
|5||0.2||0.2||2||12.6||20
+
-
|}
+
-
incubate at 37℃, for 1.5 hours<br><br>
+
-
====PCR Purification====
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!Kil||pSB1C3||Ligation High||Total
+
-
|-
+
-
|5||1||3||9
+
-
|}
+
-
at 4℃, for overnight
+
-
 
+
-
==March 2==
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!Kil||pSB1C3||Ligation High Ver.2||Total
+
-
|-
+
-
|5||1||3||9
+
-
|}
+
-
{| class="wikitable"
+
-
!tatABCD||pSB1C3||Ligation||Total
+
-
|-
+
-
|10||1||5||16
+
-
|}
+
-
at 16℃ for 1 hour<br><br>
+
-
====Transformation====
+
-
{| class="wikitable"
+
-
!Kil||Kil(3/1,Ligation)||tatABCD||competet cell||Total
+
-
|-
+
-
|1||0||0||10||11
+
-
|-
+
-
|0||1||0||10||11
+
-
|-
+
-
|0||0||1||10||11
+
-
|}
+
-
====PCR====
+
-
{| class="wikitable"
+
-
!Quick Taq||primer-r||primer-f||template||MilliQ||Total
+
-
|-
+
-
|25||1||1||0.5||22.5||50
+
-
|}
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis030227.JPG|200px|thumb|right]]
+
-
<br><br><br><br>
+
-
 
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!pSB3C5-5||EcoR1||Pst1||BufferH||BSA||MilliQ||Total
+
-
|-
+
-
|20||0.5||0.5||3||0.5||5.5||30
+
-
|}
+
-
at 37℃, for 2 hour<br><br>
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis030225.JPG|200px|thumb|right]]
+
-
1. 1kb ladder 2µL<br>
+
-
2. pSB3C5 5µL + 6×Loading Buffer 1µL<br>
+
-
・product of gel extraction(about 2700bp)<br>
+
-
-30.9µg/mL<br><br>
+
-
====Restrictive Digestion====
+
-
GFP1 ,2 ,3
+
-
{| class="wikitable"
+
-
!GFP||EcoR1||Pst1||Buffer||BSA||MilliQ||Total
+
-
|-
+
-
|10||0.5||0.5||3||0.5||15.5||30
+
-
|}
+
-
at 37℃, for 2.5 hours<br><br>
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!DT||EcoR1||Xba1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|10||0.2||0.2||3||0.3||16.3||30
+
-
|}
+
-
{| class="wikitable"
+
-
!Constitutive Promoter||Spe1||Pst1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|15||0.2||0.2||3||0.3||11.3||30
+
-
|}
+
-
at 37℃, 2 hours<br>
+
-
*J23117-1:135ng/µL, J23107-1:115ng/µL<br>
+
-
*DT3→PCR Purification<br>
+
-
*Promoter→Gel Extraction<br><br>
+
-
====Checking TMAO====
+
-
{| class="wikitable"
+
-
!something seems to be TMAO||Buffer2||EcoR1||MilliQ||Total
+
-
|-
+
-
|10||2||0.5||7.5||20
+
-
|}
+
-
at 37℃, for 1 hour<br><br>
+
-
 
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis030226.JPG|200px|thumb|right]]
+
-
1. 1kb ladder<br>
+
-
2. GFP1 that had been cut by restriction enzyme<br>
+
-
3. GFP2 that had been cut by restriction enzyme<br>
+
-
4. GFP3 that had been cut by restriction enzyme<br>
+
-
5. GFP1<br>
+
-
6. GFP2<br>
+
-
7. GFP3<br>
+
-
8. TMAO (control)<br>
+
-
9. TMAO (EcoR1)<br>
+
-
10. DT (control)<br>
+
-
11. DT (EcoR1, Xba1)<br>
+
-
12. 1kb ladder<br><br>
+
-
'''Checking tatABCD'''<br>
+
-
{| class="wikitable"
+
-
!Quick Taq||primer-f||primer-r||MilliQ||Total
+
-
|-
+
-
|25||1||1|||23||50
+
-
|}
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis030228.JPG|200px|thumb|right]]
+
-
<br><br><br><br><br><br><br><br>
+
-
 
+
-
==March 3==
+
-
====PCR====
+
-
{| class="wikitable" style="text-align: right"
+
-
! ||template||buffer||dNTPs||MgSO4||VF||VR||KOD plus||MilliQ||Total
+
-
 
+
-
|-
+
-
|1||1||5||5||3||1.5||1.5||1||32||50
+
-
|-
+
-
|2||2||5||5||3||1.5||1.5||1||31||50
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
50℃, 30sec<br>
+
-
68℃, 1min<br>
+
-
→30cycles<br><br>
+
-
====Miniprep====
+
-
14.2µg/mL, 15.0µg/mL
+
-
 
+
-
==March 4==
+
-
====Sequence of tatABCD====
+
-
{| class="wikitable" style="text-align: right;"
+
-
!Quick Taq||primer-f||promer-r sequence||template||MilliQ||Total
+
-
|-
+
-
|25||1||1||1||23||50
+
-
|}
+
-
{| class="wikitable" style="text-align: right;"
+
-
!Quick Taq||primer-f sequence||primer-r||template||MilliQ||Total
+
-
|-
+
-
|25||1||1||1||23||50
+
-
|}
+
-
====Colony PCR of TMAO====
+
-
{| class="wikitable" style="text-align: right;"
+
-
!buffer||dNTPs||NgSO4||primer-f||primerr-r||KOD plus||MilliQ||Total
+
-
|-
+
-
|5||5||4||1.5||1.5||1||32||50
+
-
|}
+
-
→ethanol precipitation<br>
+
-
94℃, 2min<br>
+
-
94℃, 30sec<br>
+
-
55℃, 30sec<br>
+
-
68℃, 2.5min<br>
+
-
→25cycles<br><br>
+
-
====Electrophoresis====
+
-
[[File:34 percent 20~2.JPG|200px|thumb|right]]
+
-
1. 1kb ladder<br>
+
-
2. tatABCD1<br>
+
-
3. tatABCD2<br>
+
-
4. TMAO<br>
+
-
5. 1kb ladder<br><br>
+
-
 
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!TMAO||EcoR1||BufferH||BSA||MilliQ||Total
+
-
|-
+
-
|10||0.2||3||0.3||16.5||30
+
-
|}
+
-
{|class="wikitable"
+
-
!TMAO||Xba1||Pst1||BudderM||BSA||MilliQ||Total
+
-
|-
+
-
|10||0.2||0.2||3||0.3||16.3||30
+
-
|}
+
-
at 37℃ for 1 hour<br><br>
+
-
====Electrophoresis====
+
-
[[File:34 percent 20~1.JPG|200px|thumb|right]]
+
-
<br><br><br><br><br><br><br>
+
-
 
+
-
====Transformation====
+
-
{|class="wikitable"
+
-
!pSB1C3||competent cell(made at 2/8)||Total
+
-
|-
+
-
|5||100||105
+
-
|}
+
-
==March 5==
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!pSB1C3(Xba1, Spe1)||Pst1||BufferH||BSA||MilliQ||Total
+
-
|-
+
-
|10||0.2||2||0.2||7.6||20
+
-
|-
+
-
!pSB1C3(Xba1, Spe1)||EcoR1||BufferH||BSA||MilliQ||Total
+
-
|-
+
-
|10||0.2||2||0.2||7.6||20
+
-
|}
+
-
at 37℃ for 1 hour<br>
+
-
→Then we did ethanol precipitation<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!Kil(EcoR1, Spe1)||pSB1C3(EcoR1)||Ligation High||Total
+
-
|-
+
-
|5||1||3||9
+
-
|}
+
-
at 16℃ for 1 hour<br><br>
+
-
====Transformation====
+
-
{| class="wikitable"
+
-
!Kil||competent cell||Total
+
-
|-
+
-
|1||10||11
+
-
|}
+
-
We used commercially available competent cells in this time.<br><br>
+
-
====PCR====
+
-
TMAO<br>
+
-
{| class="wikitable"
+
-
!buffer||dNTPs||MgSO4||Primer-f||Primer-r||Template||KOD plus||MilliQ||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||1||32||50
+
-
|}
+
-
====Electrophoresis====
+
-
[[File:35percent20~2.JPG|200px|thumb|right]]<br><br><br><br><br><br>
+
-
 
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
! ||LacP||pSB3C5||EcoR1||Pst1||BufferH||BSA||MilliQ||Total
+
-
|-
+
-
|1||20||0||0.5||0.5||3||0.5||5.5||30
+
-
|-
+
-
|2||0||20||0.5||0.5||3||0.5||5.5||30
+
-
|}
+
-
at 37℃  for 1 hour<br>
+
-
1→Ethanol precipitation 45.4µg/mL<br>
+
-
2→Gel extraction 38.7µg/mL<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!LacP||pSB3C5||Ligation High||Total
+
-
|-
+
-
|10||2||6||18
+
-
|}
+
-
at 4℃ for overnight<br><br>
+
-
====Transformation====
+
-
{| class="wikitable"
+
-
!LacP+pSB3C5||competent cell||Total
+
-
|-
+
-
|1||10||11
+
-
|}
+
-
on ice for 30 mins.<br>
+
-
heat shock at 42℃ for 60secs<br>
+
-
on ice for 2 mins.<br>
+
-
After we incubate with 200µL of SOC culture for 1 hour, we did plating on LB culture with CP<br><br>
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!GFP Plasmid||EcoR1||Spe1||Buffer2||BSA||MilliQ||Total
+
-
|-
+
-
|10||0.5||0.5||3||0.5||15.5||30
+
-
|}
+
-
at 37℃ for 2 hours
+
-
====Electrophoresis====
+
-
1. Ladder 2µL<br>
+
-
2. GFP Plasmid (already restricted) 2µL + Loading Dye 2µL + MilliQ 8µL<br>
+
-
3. Ladder 2µL<br>
+
-
====PCR====
+
-
torA signal and pspA<br>
+
-
We did Colony PCR to pspA<br>
+
-
{| class="wikitable"
+
-
!Buffer||dNTPs||MgSO4||Primer-f||Primer-r||template(TMAO)||KOD plus||MilliQ||Total
+
-
|-
+
-
|2.5||2.5||1.5||0.75||0.75||0.5||0.5||16||25
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
60℃, 30sec<br>
+
-
68℃, 30sec<br>
+
-
 
+
-
====electrophoresis====
+
-
1. 100bp Ladder<br>
+
-
2. torA signal (272bp)<br>
+
-
3. pspA (969bp)<br>
+
-
4. 100bp Ladder<br>
+
-
==March 6==
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!GFP||EcoR1||Spe1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|12||0.5||0.5||3||0.5||13.5||30
+
-
|-
+
-
|}
+
-
We did incubate at 37℃ for 1.5hours.<br>
+
-
And we did gel extraction on 3/7 and get 40.0μg/mL GFP.<br><br>
+
-
====PCR====
+
-
{| class="wikitable"
+
-
!buffer||dNTPs||MgSO4||Primer-f||Primer-r||template||KOD plus neo||MilliQ||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||0.5||1||32.5||50
+
-
|}
+
-
94℃ 2min<br>
+
-
98℃ 10sec<br>
+
-
60℃ 30sec<br>
+
-
68℃ (torA 10sec / pspA 30sec)    25 cycles<br>
+
-
We used product of PCR on 3/5 of torA and pspA as template.<br><br>
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!Kil||EcoR1||Spe1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|10||0.3||0.3||3||0.3||16.1||30
+
-
|}
+
-
{| class="wikitable"
+
-
!DT||EcoR1||Xba1||Buffer2||BSA||MilliQ||Total
+
-
|-
+
-
|10||0.5||0.5||3||0.5||15.5||30
+
-
|}
+
-
at 37℃ for 2 hours<br>
+
-
→ purification 37.7ng/μL<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!Kil||pSB1C3||Ligation High Ver.2||Total
+
-
|-
+
-
|4||2||3||9
+
-
|}
+
-
*Kil : 350fmol<br>
+
-
*pSB1C3 : 29fmol<br>
+
-
at 16℃ for overnight<br>
+
-
==March 7==
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis0307.JPG|200px|thumb|right]]<br>
+
-
1. 1kb ladder 2µL<br>
+
-
2. pspA (PCR product)2.5µL + Loading Dye 0.5µL<br>
+
-
3. GFP 30µL + Loading Dye 6µL<br>
+
-
4. 1kb ladder 2µL<br>
+
-
*The GFP was gel extracted on 3/6 and concentrated by Vacuum in 150µg/mL<br><br>
+
-
 
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!VectorDNA||GFP||Ligation High Ver.2||Total
+
-
|-
+
-
|5||15||10||30
+
-
|}
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!torA||EcoR1||Spe1||bufferM||BSA||MilliQ||Total
+
-
|-
+
-
|10||0.3||0.3||3||0.3||16.1||30
+
-
|}
+
-
{| class="wikitable"
+
-
!pspA||EcoR1||Spe1||bufferM||BSA||MilliQ||Total
+
-
|-
+
-
|5||0.3||0.3||3||0.3||21.1||30
+
-
|}
+
-
at 37℃ for 1.5 hours<br><br>
+
-
====Purification====
+
-
torA→31.8ng/µL<br>
+
-
pspA→49.3ng/µL<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!torA||pSB1C3||Ligation High Ver.2||Total
+
-
|-
+
-
|3||3||3||9
+
-
|}
+
-
{| class="wikitable"
+
-
!pspA||pSB1C3||Ligation High Ver.2||Total
+
-
|-
+
-
|4||2||3||9
+
-
|}
+
-
at 4℃, for overnight<br>
+
-
*torA→31.8ng/µL×3µL=95.4ng=0.529pmol<br>
+
-
*pSB1C3→19.4ng/µL×3µL=58.2ng=0.042pmol<br>
+
-
*pspA→49.3ng/µL×4µL=197.2ng=0.308pmol<br>
+
-
*pSB1C3→19.4ng/µL×2µL=38.8ng=0.029pmol<br>
+
-
==March 8==
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!pSB4K5||EcoR1||Spe1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|20||0.2||0.2||3||0.2||6.4||30
+
-
|-
+
-
|}
+
-
at 37℃ for 1 hour.<br>
+
-
→Purification : 36.6ng/µL<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!Kil||pSB4K5||Ligation High Ver.2||Total
+
-
|-
+
-
|10||1||5||16
+
-
|}
+
-
at 4℃ for overnight<br>
+
-
*Kil→37.7ng/µL×10µL=377ng=879fmol<br>
+
-
*pSB4K5→36.6ng/µL×1µL=36.6ng=86fmol<br><br>
+
-
====Liquid culture====
+
-
LacP + pSB3C5 -1, 2<br><br>
+
-
====Transformation====
+
-
{| class="wikitable"
+
-
!torA||pspA||competent cell||Total
+
-
|-
+
-
|1||0||10||11
+
-
|-
+
-
|0||1||10||11
+
-
|}
+
-
We use commercially available competent cells in this time.<br>
+
-
==March 9==
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!tatABCD||Xba1||Pst1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|10||0.2||0.2||3||0.3||16.3||30
+
-
|}
+
-
at 37℃ for 1hour<br>
+
-
→purification : 75.0μg/mL    (1.11  260/280 , 0.81  260/230)<br><br>
+
-
 
+
-
====Miniprep====
+
-
LacP + pSB3C5 1    80.5μg/mL    (1.78  260/280 , 2.00  260/230)<br>
+
-
LacP + pSB3C5 2    107.2μg/mL  (1.83  260/280 , 1.90  260/230)<br><br>
+
-
====Colony PCR====
+
-
{| class="wikitable"
+
-
!Quick Taq||VF||VR||MilliQ||Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
94℃ 2min<br>
+
-
94℃ 30sec<br>
+
-
55℃ 30sec<br>
+
-
68℃ 6sec<br>
+
-
25cycles<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!tatABCD||constP J23107||Ligation High Ver.2||Total
+
-
|-
+
-
|5||1||3||9
+
-
|}
+
-
tatABCD : 227fmol<br>
+
-
constP J23107 : 21fmol<br><br>
+
-
====Transformation====
+
-
{| class="wikitable"
+
-
! ||pspA||torA||Kil||competent cell
+
-
|-
+
-
|1||1||0||0||10
+
-
|-
+
-
|2||0||1||0||10
+
-
|-
+
-
|3||0||0||1||10
+
-
|}
+
-
====Miniprep====
+
-
4mL of plusgrow which had been cultured for overnight.<br>
+
-
pSB4K5 : 80.5μg/mL<br>
+
-
==March 10==
+
-
====Screening PCR====
+
-
{| class="wikitable"
+
-
!Quick Taq||VF||VR||MilliQ||Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
[[File:Electrophoresis0310.JPG|200px|thumb|right]]
+
-
Then we did electrophoresis to confirm.<br>
+
-
1. 1kb ladder<br>
+
-
2. kil (649bp)<br>
+
-
3. 4. 5. pspA (969bp)<br>
+
-
6. 1kb ladder<br><br>
+
-
1. 100bp ladder<br>
+
-
2. 3. 4. torA signal<br>
+
-
 
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!LacP-pSB3C5||Spe1||Pst1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|10||0.2||0.2||2||0.2||7.4||20
+
-
|}
+
-
for 2.5 hours at 37℃<br>
+
-
→purification  29.0ng/μL<br>
+
-
{| class="wikitable"
+
-
!torA||Xba1||Pst1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|10||0.2||0.2||2||0.2||7.4||20
+
-
|}
+
-
for 2.5 hours at 37℃<br>
+
-
→purification  91.8ng/μL<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!torA||LacP-pSB3C5||Ligation High Ver.2||Total
+
-
|-
+
-
|4||2||3||9
+
-
|}
+
-
torA : 929fmol<br>
+
-
LacP-pSB3C5 : 284fmol<br>
+
-
for overnight at 4℃
+
-
==March 11==
+
-
====Miniprep====
+
-
We used 3μL of plus grow that we had cultured for overnight.<br>
+
-
torA : 62.8ng/μL<br><br>
+
-
====Screening PCR====
+
-
{| class="wikitable"
+
-
!Quick Taq||VF||VR||MilliQ||Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis031101.JPG|200px|thumb|right]]
+
-
[[File:Electrophoresis031102.JPG|200px|thumb|right]]
+
-
1. 1kb ladder<br>
+
-
2〜11. pspA (969bp)<br>
+
-
12. 1kb ladder<br>
+
-
<br>The results were shown as photograph in the right.<br>
+
-
<br>It seemed that there were shorter sample than expected sample, so we did electrophoresis with pspA which was product of PCR and pspA which had already cut with EcoR1 and Spe1.<br><br><br><br><br><br>
+
-
1.1kb ladder<br>
+
-
2. pspA (PCR product)<br>
+
-
3, pspA (Eco, Spe)<br>
+
-
4〜6, pspA (colony PCR)<br>
+
-
7,1kb ladder<br>
+
-
<br>The results were shown as photograph in the right.<br><br>
+
-
 
+
-
==March 12==
+
-
====Transformation====
+
-
{| class="wikitable"
+
-
!DT(1ng/μL)||DT(0.1ng/μL)||Kil||LacP-torA||MilliQ||competent cell||Total
+
-
|-
+
-
|1||0||0||0||0||20||21
+
-
|-
+
-
|0||1||0||0||0||20||21
+
-
|-
+
-
|0||0||5||0||0||50||51
+
-
|-
+
-
|0||0||0||5||0||50||51
+
-
|-
+
-
|0||0||0||0||1||20||21
+
-
|}
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!pspA||EcoR1||Spe1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|10||0.5||0.5||3||0.3||15.7||30
+
-
|}
+
-
at 37℃ for 4 hours<br>
+
-
→ We did purification and got 48.3ng/μL pspA.<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
! |pspA||pSB1C3||MilliQ||Ligation High Ver.2||Total
+
-
|-
+
-
|1|4||2||0||3||9
+
-
|-
+
-
|2|2||2||0||2||6
+
-
|-
+
-
|3|0||2||2||2||6
+
-
|}
+
-
==March 13==
+
-
====Miniprep====
+
-
pSB1C3  74.2µg/mL  1.65 (260/280)  1.31 (260/230)
+
-
==March 14==
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!pSB1C3||EcoR1||Spe1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|20||0.2||0.2||4||0.4||15.2||40
+
-
|}
+
-
We did gel extraction and got 47.2ng/µL pSB1C3 but we did not cut out RFP.<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!torA||pSB1C3||Ligation High Ver.2||Total
+
-
|-
+
-
|4||2||3||9
+
-
|-
+
-
!MilliQ||pSB1C3||Ligation High Ver.2||Total
+
-
|-
+
-
|4||2||3||9
+
-
|}
+
-
at 16℃, for 1 hour<br>
+
-
*torA : 0.707pmol<br>
+
-
*pSB1C3 : 0.068pmol<br><br>
+
-
====Liquid culture====
+
-
We cultured LacP-pSB3C5 1,2,3,4,5 (CP tolerance)on culture with Amp.<br>
+
-
→Only 4 which did not be cultured succeeded.
+
-
==March 15==
+
-
====Liquid culture====
+
-
We cultured LacP-pSB3C5 6,7,8,9,10 on culture with ampicillin.<br>
+
-
→6,8,9,10 were succeeded.<br><br>
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!GFP||EcoR1||Spe1||Buffer2||BSA||MilliQ||Total
+
-
|-
+
-
|10||0.5||0.5||3||0.5||15.5||30
+
-
|}
+
-
{| class="wikitable"
+
-
!DT||EcoR1||Xba1||Buffer2||BSA||MilliQ||Total
+
-
|-
+
-
|10||0.5||0.5||3||0.5||15.5||30
+
-
|}
+
-
for 2 hours at 37℃.<br><br>
+
-
====Miniprep====
+
-
pspA (pSB1C3)  40.5ng/µL<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!pspA||DT||Ligation High Ver.2||Total
+
-
|-
+
-
|5||1.5||3||9.5
+
-
|}
+
-
*pspA : 385fmol<br>
+
-
*DT : 36fmol<br><br>
+
-
====Transformation====
+
-
{| class="wikitable"
+
-
!J23107-tatABCD||DT (0.1ng/µL)||DT (0.01ng/µL)||pspA-DT||competent cells on 3/15||Total
+
-
|-
+
-
|2||0||0||0||20||22
+
-
|-
+
-
|0||2||0||0||20||22
+
-
|-
+
-
|0||0||2||0||20||22
+
-
|-
+
-
|0||0||0||2||20||22
+
-
|}
+
-
====Screening PCR====
+
-
Kil, pspA and torA<br>
+
-
{| class="wikitable"
+
-
!Quick Taq||Primer-r||Primer-f||MilliQ||Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
 
+
-
==March 16==
+
-
====Miniprep====
+
-
torA (pSB1C3)  68.8ng/µL<br>
+
-
Kil (pSB4K5)  92.7ng/µL<br>
+
-
torA was red for some reason. We do not know why.<br><br>
+
-
====Colony PCR====
+
-
{| class="wikitable"
+
-
!Quick Taq||Primer-r||Primer-f||MilliQ||Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
94℃, 2min<br>
+
-
94℃, 30sec<br>
+
-
55℃, 30sec<br>
+
-
68℃, 6sec<br>
+
-
→25cycles<br><br>
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis0316.JPG|200px|thumb|right]]
+
-
The results were shown as photograph in the right.<br><br><br><br><br>
+
-
 
+
-
====Checking Transformation Efficiency====
+
-
competent cells that were made on March 15.<br>
+
-
DNA : 0.02ng → 668 colonies  Transformation Efficiency : 3.3×10^7<br>
+
-
DNA : 0.2ng → 1739 colonies  Transformation Efficiency : 8.7×10^6<br><br>
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!pSB1C3||EcoR1||Spe1||BSA||BufferM||BufferH||MilliQ||Total
+
-
|-
+
-
|20||0.2||0.2||0.3||3||0||6.3||30
+
-
|-
+
-
|5||0.2||0||0.2||0||2||12.6||20
+
-
|}
+
-
at 37℃, for 2 hours.<br>
+
-
We did gel extraction for product with EcoR1, Spe1. We got 46.1ng/µL pSB1C3.<br>
+
-
{| class="wikitable"
+
-
!Kil(pSB4K5)||EcoR1||Pst1||BSA||BufferH||MilliQ||Total
+
-
|-
+
-
|5||0.2||0.2||0.2||2||12.4||20
+
-
|-
+
-
|5||0.2||0||0.2||2||12.6||20
+
-
|}
+
-
for overnight at 37℃.<br>
+
-
We did this to confirm.<br>
+
-
{| class="wikitable"
+
-
!pspA (pSB1C3)||EcoR1||Pst1||BSA||BufferH||MilliQ||Total
+
-
|-
+
-
|5||0.2||0.2||0.2||2||12.4||20
+
-
|-
+
-
|5||0.2||0||0.2||2||12.6||20
+
-
|}
+
-
for overnight at 37℃.<br>
+
-
We did this to confirm.<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!torA||pSB1C3||Ligation High Ver.2||Total
+
-
|-
+
-
|4||2||3||9
+
-
|}
+
-
{| class="wikitable"
+
-
!MilliQ||pSB1C3||Ligation High Ver.2||Total
+
-
|-
+
-
|4||2||3||9
+
-
|}
+
-
at 16℃, for overnight<br>
+
-
*torA→767fmol<br>
+
-
*pSB1C3→68fmol<br>
+
-
==March 17==
+
-
====Miniprep====
+
-
J23107-tatABCD  72.7ng/µL<br>
+
-
pspA-DT  50.5ng/µL<br><br>
+
-
====Checking the Insert====
+
-
{| class="wikitable"
+
-
!J21037-tatABCD||EcoR1||Pst1||BSA||BufferH||MilliQ||Total
+
-
|-
+
-
|5||0.2||0.2||0.2||2||12.4||20
+
-
|-
+
-
|5||0.2||0||0.2||2||12.6||20
+
-
|}
+
-
Success.
+
-
{| class="wikitable"
+
-
!pspA-DT||EcoR1||Pst1||BSA||BufferH||MilliQ||Total
+
-
|-
+
-
|5||0.2||0.2||0.2||2||12.4||20
+
-
|-
+
-
|5||0.2||0||0.2||2||12.6||20
+
-
|}
+
-
Failed.
+
-
==March 19==
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!DT||EcoR1||Xba1||BSA||BufferM||MilliQ||Total
+
-
|-
+
-
|10||0.2||0.2||0.3||3||16.3||30
+
-
|}
+
-
We did Gel extraction and got 17.2ng/µL of DT.<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!Kil||DT||Ligation High Ver.2||Total
+
-
|-
+
-
|10||2||6||18
+
-
|}
+
-
We did this for an hour at 16℃.<br><br>
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!GFP||EcoR1||Spe1||BSA||BufferM||MilliQ||Total
+
-
|-
+
-
|10||0.5||0.5||0.5||3||15.5||30
+
-
|}
+
-
We did this for 4 hours at 37℃<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!pspA||DT||Ligation High Ver.2||Total
+
-
|-
+
-
|5||5||5||15
+
-
|}
+
-
{| class="wikitable"
+
-
!pspA||pSB1C3||Ligation High Ver.2||Total
+
-
|-
+
-
|4||1||3||8
+
-
|}
+
-
We did these for an hour at 16℃.<br>
+
-
*pspA (5µL)→377fmol<br>
+
-
*DT→39fmol<br>
+
-
*pspA (4µL)→339fmol<br>
+
-
*pSB1C3→34fmol<br><br>
+
-
====Transformation====
+
-
pspA-DT, pspA (pSB1C3), torA (pSB1C3) and GFP-DT
+
-
==March 20==
+
-
====Screaning PCR====
+
-
{| class="wikitable"
+
-
!Quick Taq||Primer-R||Primer-F||MilliQ||Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
*pspA→○<br>
+
-
*pspA-DT→○<br>
+
-
*GFP-DT→○<br>
+
-
*torA→×<br>
+
-
*Kil-DT 6 of 8 sumples→○<br>
+
-
{| class="wikitable"
+
-
!Quick Taq||VR||VF||MilliQ||Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
*pspA→○<br>
+
-
*pspA-DT→×<br><br>
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!torA||EcoR1||Spe1||BSA||BufferM||MilliQ||Total
+
-
|-
+
-
|10||0.3||0.3||0.3||3||16.1||30
+
-
|}
+
-
{| class="wikitable"
+
-
!DT||EcoR1||Xba1||BSA||BufferM||MilliQ||Total
+
-
|-
+
-
|10||0.2||0.2||0.3||3||16.3||30
+
-
|}
+
-
We did this for overnight at 37℃. And we did purification.<br>
+
-
torA  34.2ng/µL<br>
+
-
DT  28.0ng/µL<br><br>
+
-
==March 21==
+
-
====Miniprep====
+
-
GFP-DT-1 : 77.7ng/µL<br>
+
-
GFP-DT-2 : 67.6ng/µL<br><br>
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!pSB1C3||EcoR1||Spe1||BSA||BufferM||MilliQ||Total
+
-
|-
+
-
|10||0.2||0.2||0.3||3||16.3||30
+
-
|-
+
-
|5||0.2||0||0.2||2||12.6||20
+
-
|}
+
-
We did this for 3 hours at 37℃, and then we did gel extraction. We got 25.1ng/µL pSB1C3<br><br>
+
-
{| class="wikitable"
+
-
!GFP-DT||EcoR1||Pst1||Xba1||BSA||BufferM||MilliQ||Total
+
-
|-
+
-
|5||0.2||0.2||0||0.2||2||12.4||20
+
-
|-
+
-
|5||0.2||0||0||0.2||2||12.6||20
+
-
|-
+
-
|10||0.2||0||0.2||0.3||3||16.3||30
+
-
|}
+
-
We did this for 3 hours at 37℃, and then we did Purification. We got 30.7ng/µL GFP-DT.<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
! ||torA||pSB1C3||pspA||DT||GFP-DT||Ligation High Ver.2||Total
+
-
|-
+
-
|1||4||1||0||0||0||3||8
+
-
|-
+
-
|2||0||1||7||0||0||4||12
+
-
|-
+
-
|3||0||0||5||3||0||4||12
+
-
|-
+
-
|4||3||0||0||0||5||4||12
+
-
|}
+
-
We did this for an hour at16℃.
+
-
==March 22==
+
-
====PCR====
+
-
We did PCR to amplify torA_signal that was product of PCR at March 5 with redesigned primers.<br>
+
-
{| class="wikitable"
+
-
!Buffer||dNTPs||MgSO4||Primer-F||Primer-R||Template||MilliQ||KOD plus neo||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||0.5||32.5||1||50
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
60℃, 30sec<br>
+
-
68℃, 10sec<br>
+
-
→30cycles<br>
+
-
→Purification  110.7ng/µL<br><br>
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!torA||EcoR1||Spe1||BSA||BufferM||MilliQ||Total
+
-
|-
+
-
|10||0.2||0.2||0.3||3||16.3||30
+
-
|}
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
! ||torA||pSSB1C3||pspA||DT||GFP-DT||Ligation high||Total
+
-
|-
+
-
|1||4||3||0||0||0||4||11
+
-
|-
+
-
|2||3||0||0||0||3||3||9
+
-
|-
+
-
|3||0||0||5||5||0||5||15
+
-
|}
+
-
*torA (4µL)→512fmol<br>
+
-
*pSB1C3→54fmol<br>
+
-
*torA (3µL)→384fmol<br>
+
-
*GFP-DT→36fmol<br>
+
-
*pspA→377fmol<br>
+
-
*DT→65fmol<br><br>
+
-
 
+
-
==March 23==
+
-
====Screening PCR====
+
-
torA (pSB1C3), torA-GFP-DT and pspA-DT<br>
+
-
{| class="wikitable"
+
-
!Quick Taq||VF||VR||MilliQ||Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
E.coli in liquid culture that had pspA(pSB1C3) also expressed RFP.<br><br>
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!pspA||EcoR1||Spe1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|5||0.3||0.3||3||0.3||21.1||30
+
-
|}
+
-
And then we did ethanol precipitation<br><br>
+
-
====Ethanol precipitation====
+
-
pspA 11.5ng/µL.<br><br>
+
-
====Miniprep====
+
-
LacP+pSB3C5-8  77.9µg/mL<br>
+
-
LacP+pSB3C5-10  69.0µg/mL<br>
+
-
Kil+DT-4  58.0µg/mL<br>
+
-
Kil+DT-7  15.2µg/mL<br><br>
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!LacP+pSB3C5-8||Spe1||Pst1||Buffer2||BSA||MilliQ||Total
+
-
|-
+
-
|20||0.5||0.5||3||0.5||5.5||30
+
-
|}
+
-
{| class="wikitable"
+
-
!Kil+DT-4||Xba1||Pst1||Buffer2||BSA||MilliQ||Total
+
-
|-
+
-
|20||0.5||0.5||3||0.5||5.5||30
+
-
|}
+
-
at 37℃ for 1.5 hours<br>
+
-
And then we did Gel extraction.<br><br>
+
-
====Gel Extraction====
+
-
LacP+pSB3C5-8  40.4µg/mL<br>
+
-
Kil+DT-4  26.8µg/mL<br>
+
-
 
+
-
==March 26==
+
-
====Miniprep====
+
-
torA(pSB1C3) 18.5[ng/µL]<br>
+
-
torA-GFP-DT 20.0[ng/µL]<br><br>
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!torA(pSB1C3)||EcoR1||Pst1||BufferH||BSA||MilliQ||Total
+
-
|-
+
-
|5||0.2||0.2||2||0.2||12.4||20
+
-
|-
+
-
|5||0.2||0||2||0.2||12.6||20
+
-
|}
+
-
{| class="wikitable"
+
-
!torA-GFP-DT||EcoR1||Xba1||Pst1||BufferH||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|5||0.2||0||0.2||2||0||0.2||12.4||20
+
-
|-
+
-
|5||0.2||0||0||2||0||0.2||12.6||20
+
-
|-
+
-
|20||0||0.2||0.2||0||3||0.3||6.3||30
+
-
|}
+
-
We did Gel extraction and then got ??? 28.7[ng/µL]<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!LacP (pSB3C5)||torA-GFP-DT||Ligation High Ver.2||Total
+
-
|-
+
-
|1||5||3||9
+
-
|}
+
-
*LacP : 22fmol<br>
+
-
*torA-GFP-DT : 197fmol<br>
+
-
{| class="wikitable"
+
-
!pspA||pSB1C3||Ligation High Ver.2||Total
+
-
|-
+
-
|10||1||5||16
+
-
|}
+
-
{| class="wikitable"
+
-
!pspA||DT||Ligation High Ver.2||Total
+
-
|-
+
-
|10||1||5||16
+
-
|}
+
-
*pspA : 180fmol<br>
+
-
*pSB1C3 : 18fmol<br>
+
-
*DT : 16fmol<br>
+
-
{| class="wikitable"
+
-
!LacP(pSB3C5)||Kil-DT||Ligation High Ver.2||Total
+
-
|-
+
-
|1||5||3||9
+
-
|}
+
-
for 2 hours at 16℃<br><br>
+
-
====Transformation====
+
-
{| class="wikitable"
+
-
!LacP-Kil-DT||competent cell||Total
+
-
|-
+
-
|1||10||11
+
-
|}
+
-
 
+
-
==March 27==
+
-
====Miniprep====
+
-
We retryed miniprep of torA(pSB1C3).<br>
+
-
We got torA and its concentration was 39.3[ng/µL].<br><br>
+
-
====Transformation====
+
-
{| class="wikitable" 
+
-
!Name||Well||Sample||Competent Cells||Total||Plate||Colony
+
-
|-
+
-
|BBa_K117004 ||14J(2011 plate2)||5||20||?||?||?
+
-
|}
+
-
We added 100[µL] of culture medium before we started culturing the E.coli.<br><br>
+
-
====Screening PCR====
+
-
{| class="wikitable"
+
-
!Quick Taq||VF2||VR||MilliQ||Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
LacP-torA-GFP-DT 1, 3, 5, 6, 8, 9 was successful.<br>
+
-
pspA-DT was failed.<br>
+
-
E.coli that had pspA(pSB1C3) did not make any colony.<br>
+
-
LacP-Kil-DT 1,2,3,4,5,6,7,8 was failed.<br><br>
+
-
====Liquid culture====
+
-
LacP-torA-GFP-DT<br>
+
-
 
+
-
==July 23==
+
-
====Transformation====
+
-
{|class="wikitable"
+
-
!DT(64.4ng/μl)||Competent cell||Plating in SOC medium||Total
+
-
|-
+
-
|1||20||100||121
+
-
|}
+
-
 
+
-
==July 24==
+
-
====Plating in SOC medium====
+
-
 
+
-
==July 25==
+
-
====Transformation====
+
-
*BBa_J23113 from iGEM Kit
+
-
*LacP from iGEM Kit
+
-
 
+
-
==July 27==
+
-
====Restrictive Digestion====
+
-
{|class="wikitable"
+
-
!Kil +DT (44.0 ng/μl)||10xBuffer2||BSA||Xbal||MilliQ||Total
+
-
|-
+
-
|13.6||3.0||0.5||0.5||11.9||30.0
+
-
|}
+
-
{|class="wikitable"
+
-
!LacP(28.7ng/μl)||10xBuffer2 ||BSA||Spe1||Pst1||Total
+
-
|-
+
-
|20.9||3.0||0.5||0.5||4.6||30.0
+
-
|}
+
-
 
+
-
==July 30==
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!kil+DT(Xba1,Pst1)||LacP(Spe1,Pst1)||Ligation High ||Total
+
-
|-
+
-
|30||6||36||72
+
-
|}
+
-
 
+
-
==July 31==
+
-
====Restriction Enzyme Processing ====
+
-
{| class="wikitable"
+
-
!torA-GFP+DT||10×M Buffer||BSA||MilliQ||Xba1||Pst1||Total
+
-
|-
+
-
|2.5||3.0||0.5||23.4||0.3||0.3||30.0
+
-
|}
+
-
====Liquid Culture====
+
-
J23113(backbone J61002)
+
-
 
+
-
==August 1==
+
-
====Restrictive Digestion====
+
-
{|class="wikitable"
+
-
!LacP+kil+DT ||BSA||10×H Buffer||EcoR1||Pst1||MilliQ||Total
+
-
|-
+
-
|5.0||0.5||3.0||0.5||0.5||20.5||30.0
+
-
|}37℃ 2h
+
-
{|class="wikitable"
+
-
!LacP middle copy ||BSA||10×H Buffer||EcoR1||Pst1||MilliQ||Total
+
-
|-
+
-
|10.0||0.5||3.0||0.5||0.5||15.5||30.0
+
-
|}37℃ 2h
+
-
====MIniprep====
+
-
J23113(backbone J61002)
+
-
====Liquid culture====
+
-
J23113(backboneJ61002)
+
-
Three test tubes of 4 mL LB medium with ampicillin
+
-
37℃ overnight
+
-
 
+
-
==August 3==
+
-
====Restrictive Digestion====
+
-
{|class="wikitable"
+
-
!pSB4K5||BSA|| 10×H Buffer||EcoR1||Pst1||MilliQ||Total
+
-
|-
+
-
|8.0||0.5||3.0||0.5||0.5||17.5||30.0
+
-
|}
+
-
{|class="wikitable"
+
-
!B0034||BSA||10×H Buffer||Spe1||Pst1||MilliQ||Total
+
-
|-
+
-
|12.0||0.5||3.0||0.5||0.5||13.5||30.0
+
-
|}
+
-
37℃ 2h
+
-
====DNA purification====
+
-
====Ligation====
+
-
{|class="wikitable"
+
-
!LacP+kil+DT||pSB4K5||ligation high||Total
+
-
|-
+
-
|15||15||15||45
+
-
|}16℃ 1h
+
-
====Miniprep====
+
-
*J23113(backbone J61002) 218.0μg/mL
+
-
*J23113(backbone J61002) 252.5μg/mL
+
-
*J23113(backbone J61002) 201.0μg/mL
+
-
*pSB3C5                  45.0μg/ml  1.60  260/280  1.80  260/230
+
-
*pSB4C5                  213.0μg/mL  1.77  260/280  4.40  260/230
+
-
 
+
-
==August 10==
+
-
====Ligation====
+
-
{|class="wikitable"
+
-
!B0034 Spe1 Pst1||GFP+DT Xba1 Pst1||ligation high||Total
+
-
|-
+
-
|2||3||4||9
+
-
|}16℃ 1h
+
-
====Transformation====
+
-
*RBS+GFP+DT
+
-
*LacP+kil+DT
+
-
*RBS(for control)
+
-
:To put 2ng DNA in 20μL competent cell and leave it on ice for 30min
+
-
:Heat shock for 60s at 42℃ 
+
-
:To leave on ice for 2min
+
-
:To spread on ampicillin LB plate
+
-
 
+
-
==August 13==
+
-
====Liquid culture====
+
-
B0034 3mL ×3    
+
-
:37℃ overnight
+
-
 
+
-
==August 14==
+
-
====Miniprep====
+
-
:B0034 0μg/mL
+
-
:B0034 235.5μg/mL
+
-
:B0034 76.5μg/mL
+
-
====Colony PCR====
+
-
LacP+kil+DT ×5
+
-
{|class="wikitable"
+
-
!2×Quick Taq||VF||VF||MilliQ||Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
25cycles <br>
+
-
pspA
+
-
{|class="wikitable"
+
-
!10×Buffer for KOD-Plus-Ver2||2mM dNTPs||25mM MgSO4||Primer PsPA  f-p||Primer PsPA  r-s||KOD-Plus-||MilliQ||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||33||50
+
-
|}
+
-
==August 15==
+
-
====Colony PCR====
+
-
pspA
+
-
{|class="wikitable"
+
-
!2×Quick Taq||pspA r-s||pspA f-p||MilliQ||Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
94℃, 2min<br>
+
-
94℃, 30sec<br>
+
-
56℃, 30sec<br>
+
-
68℃, 1min<br>
+
-
→35cycles<br><br>
+
-
 
+
-
==August 16==
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis081601.jpg|200px|right]]
+
-
[[File:0814.jpg|200px|right]]
+
-
positive control(GFP+DT)<br>
+
-
negative control(MilliQ) <br>
+
-
① ~⑥ colony PCR LacP+kil+DT<br><br><br><br><br><br>
+
-
 
+
-
①1kb ladder<br>
+
-
②Positive control (GFP+DT) <br>
+
-
③Negative control (MilliQ) <br>
+
-
④~⑨LacP+kil+DT ①~⑥<br>
+
-
⑩~⑫pspA 10μL ,6×buffer 2μL<br>
+
-
⑬1kb ladder<br>
+
-
 
+
-
==August 17==
+
-
====Colony PCR====
+
-
pspA
+
-
{|class="wikitable"
+
-
!10×Buffer for KOD-Plus-Ver2||2mM dNTPs||25mM MgSO4||Primer PsPA  f||Primer PsPA  r||KOD-Plus-||MilliQ||Total
+
-
|-
+
-
|5||3||3||1.5||1.5||1||35||50
+
-
|-
+
-
|5||3||5||1.5||1.5||1||33||50
+
-
|}
+
-
negative control(MilliQ 50μL)
+
-
94℃, 2min<br>
+
-
94℃, 15sec<br>
+
-
55℃, 30sec<br>
+
-
68℃, 1min<br>
+
-
→35cycles<br><br>
+
-
 
+
-
====Electrophoresis====
+
-
[[File:0820.JPG|200px|thumb|right]]
+
-
 
+
-
① 1kb ladder<br>
+
-
②, ③ MgSO4 3μL, pspA<br>
+
-
④, ⑤ MgSO4 5μL, pspA<br>
+
-
⑥ negative control(MilliQ)<br>
+
-
⑧ 1kb ladder<br>
+
-
<br><br><br>
+
-
 
+
-
==August 20==
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!J23113(201.0ng/μL)||Xba1||Pst1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|10||1||1||3||0.5||14.5||30
+
-
|-
+
-
|}
+
-
 
+
-
==August 21==
+
-
====Colony PCR====
+
-
pspA
+
-
{|class="wikitable"
+
-
!10×Buffer||2mM dNTPs||25mM MgSO4||Primer PsPA  f||Primer PsPA  r||KOD Plus Neo||MilliQ||Total
+
-
|-
+
-
|5||3||5||1.5||1.5||1||33||50
+
-
|-
+
-
|5||3||7||1.5||1.5||1||31||50
+
-
|-
+
-
|5||3||10||1.5||1.5||1||28||50
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
60℃, 30sec<br>
+
-
68℃, 30sec<br>
+
-
→25cycles<br><br>
+
-
====Restrictive Digestion====
+
-
{|class="wikitable"
+
-
!J23113(218ng/μL)||Spe1||Pst1||BufferH||BSA||MilliQ||Total
+
-
|-
+
-
|10||1||1||3||0.5||14.5||30
+
-
|}
+
-
{|class="wikitable"
+
-
!torA-GFP-DT(20ng/μL)||Xba1||Pst1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|5||0.6||0.6||3||0.5||20.3||30
+
-
|}
+
-
====Electrophoresis====
+
-
 
+
-
[[File:0821e.jpg|200px|thumb|right]]
+
-
 
+
-
①    1kb ladder<br>
+
-
②, ③ torA-GFP-DT(Xba1,Pst1)<br>
+
-
④, ⑤ J23113(Spe1,Pst1)<br><br><br>
+
-
 
+
-
====Transformation====
+
-
*LacP-torA-GFP-DT(Backbone pSB3C5)
+
-
*BBa_K11704(Backbone pSB1A2)<br>
+
-
 
+
-
====Electrophoresis====
+
-
 
+
-
[[File:0821r.jpg|200px|thumb|right]]
+
-
laneA-C. pspA<br>
+
-
laneD. pspA(without primer)<br><br><br><br>
+
-
 
+
-
====Colony PCR====
+
-
*pspA
+
-
{|class="wikitable"
+
-
!10×Buffer||2mM dNTPs||25mM MgSO4||Primer PsPA f||Primer PsPA r||KOD Plus Neo||MilliQ||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||33||50
+
-
|}
+
-
*negative control for pspA
+
-
{|class="wikitable"
+
-
!10×Buffer||2mM dNTPs||25mM MgSO4||Primer PsPA f-p||Primer PsPA r-s||KOD Plus Neo||MilliQ||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||33||50
+
-
|} 
+
-
*GFP-DT(positive control)
+
-
{|class="wikitable"
+
-
!10×Buffer||2mM dNTPs||25mM MgSO4||VF||VR||KOD Plus Neo||MilliQ||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||33||50
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
60℃, 30sec<br>
+
-
68℃, 30sec<br>
+
-
 
+
-
====Transformation====
+
-
*LacP-torA-GFP-DT(Backbone pSB3C5)
+
-
*J23107-tatABCD
+
-
====PCR====
+
-
kil, torA-GFP
+
-
{|class="wikitable"
+
-
!10×Buffer||2mM dNTPs||25mM MgSO4||VF||VR||KOD Plus Neo||MilliQ||DNA||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||32||1||50
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
60℃, 30sec<br>
+
-
68℃, 30sec<br>
+
-
→25cycles<br><br>
+
-
 
+
-
==August 22==
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!pSB3C5||EcoR1||Pst1||BufferH||BSA||MilliQ||Total
+
-
|-
+
-
|13.3||0.5||0.5||3||0.5||12.2||30
+
-
|}
+
-
====Gel extraction====
+
-
pSB3C5(EcoR1, Pst1)<br>
+
-
27.2μg/mL
+
-
====Transformation(the second time)====
+
-
*LacP-torA-GFP-DT(Backbone pSB3C5)
+
-
*J23107-tatABCD
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis082202.JPG|thumb|200px|right]]
+
-
See "August 21 -Colony PCR-"<br>
+
-
1. 1kb ladder<br>
+
-
2. pspA(Primer pspA f&r)<br>
+
-
3. negative control for 2<br>
+
-
4. GFP-DT<br>
+
-
5. pspA(Primer pspA f-p&r-s)<br>
+
-
6. pspA(Primer pspA f-p&r-s)<br>
+
-
7. negative control for 5&6<br>
+
-
 
+
-
====PCR====
+
-
LacP-GFP-DT(a kit of biological parts)
+
-
{|class="wikitable"
+
-
!10×Buffer||2mM dNTPs||25mM MgSO4||VF||VR||KOD Plus Neo||MilliQ||DNA||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||32||1||50
+
-
|}
+
-
====Colony PCR====
+
-
pspA
+
-
{|class="wikitable"
+
-
!10×Buffer||2mM dNTPs||25mM MgSO4||Primer pspA f||Primer pspA r||KOD Plus Neo||MilliQ||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||33||50
+
-
|}
+
-
{|class="wikitable"
+
-
!10×Buffer||2mM dNTPs||25mM MgSO4||Primer pspA f-p||Primer pspA r-s||KOD Plus Neo||MilliQ||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||33||50
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
60℃, 30sec<br>
+
-
68℃, 30sec<br>
+
-
→30cycles<br>
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis082201.jpg|200px|right]]
+
-
1. 1kb ladder<br>
+
-
2. pspA(Primer pspA f&r)<br>
+
-
3. pspA(Primer pspA f-p&r-s)<br>
+
-
4. negative control for pspA(Primer pspA f&r)<br>
+
-
5. negative control for pspA(Primer pspA f-p&r-s)<br>
+
-
 
+
-
====Transformation====
+
-
Kil
+
-
 
+
-
==August 23==
+
-
====Colony PCR====
+
-
J23107-tatABCD<br>
+
-
VF and VR were used for amplifying approximately 2800bp
+
-
{|class="wikitable"
+
-
!10×Buffer||2mM dNTPs||25mM MgSO4||VF||VR||KOD Plus Neo||MilliQ||DNA||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||32.5||0.5||50
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
60℃, 30sec<br>
+
-
68℃, 90sec<br>
+
-
→25cycles<br>
+
-
 
+
-
==August 24==
+
-
====Purification of PCR products====
+
-
pspA(Primer pspA f-p&r-s)<br>
+
-
84.1μg/mL<br>
+
-
→See "August 22 -Colony PCR-"
+
-
====Restrictive Digestion====
+
-
{|class="wikitable"
+
-
!pspA||EcoR1||Pst1||BufferH||BSA||MilliQ||Total
+
-
|-
+
-
|20||0.5||0.5||3||0.5||5.5||30
+
-
|}
+
-
{|class="wikitable"
+
-
!pspA||Xba1||Pst1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|20||0.5||0.5||3||0.5||5.5||30
+
-
|}
+
-
at 37℃ for 1h
+
-
====Gel extraction====
+
-
*pspA(EcoR1, Pst1) 30.7μg/mL
+
-
*pspA(Xba1, Pst1) 44.0μg/mL
+
-
====Restrictive Digestion====
+
-
{|class="wikitable"
+
-
!pSB1C3(82.9μg/mL)||EcoR1||Pst1||BufferH||BSA||MilliQ||Total
+
-
|-
+
-
|20||0.5||0.5||3||0.5||5.5||30
+
-
|}
+
-
→Gel extraction
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis082401.jpg|200px|thumb|right]]
+
-
1. 1kb ladder<br>
+
-
2. J23107-tatABCD →See "August 23 -PCR-"<br>
+
-
3. negative control for J23107-tatABCD<br>
+
-
4. pSB1C3(EcoR1, Pst1)<br><br>
+
-
 
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!pSB3C5(EcoR1, Pst1)||pspA(EcoR1, Pst1)||Ligation High Ver.2||Total
+
-
|-
+
-
|1||5||3||9
+
-
|}
+
-
16℃, overnight
+
-
 
+
-
==August 25==
+
-
 
+
-
+
-
 
+
-
+
-
 
+
-
 
+
-
 
+
-
 
+
-
==August 26==
+
-
 
+
-
+
-
 
+
-
+
-
 
+
-
 
+
-
 
+
-
 
+
-
==August 27==
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!pSB1C3(EcoR1, Pst1)||pspA(EcoR1, Pst1)||Ligation High Ver.2||Total
+
-
|-
+
-
|1||5||3||9
+
-
|}
+
-
16℃, overnight
+
-
 
+
-
====Colony PCR====
+
-
Kil (1-7)
+
-
{|class="wikitable"
+
-
!10×Buffer||2mM dNTPs||25mM MgSO4||VF||VR||KOD Plus Neo||MilliQ||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||33||50
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
60℃, 30sec<br>
+
-
68℃, 30sec<br>
+
-
→30cycles<br>
+
-
[[File:0827c.jpg|200px|thumb|right]]
+
-
 
+
-
====Purification of PCR products====
+
-
torA-GFP →See "August 21 -PCR-"
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!LacP(Backbone:pSB3C5)||Spe1||Pst1||BufferH||BSA||MilliQ||Total
+
-
|-
+
-
|20||0.5||0.5||3||0.5||5.5||30
+
-
|}
+
-
at 37℃ for 1h<br>
+
-
→Gel extraction<br>
+
-
{| class="wikitable"
+
-
!torA-GFP||Xba1||Pst1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|20||0.5||0.5||3||0.5||5.5||30
+
-
|}
+
-
at 37℃ for 1h<br>
+
-
→Purification
+
-
====Purification of PCR products====
+
-
J23107-tatABCD<br>
+
-
42.0μg/mL<br>
+
-
→See "August 23 -PCR-"
+
-
====Restrictive Digestion====
+
-
{|class="wikitable"
+
-
!J23107-tatABCD||Spe1||EcoR1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|20||0.5||0.5||3||0.5||5.5||30
+
-
|}
+
-
 
+
-
==August 28==
+
-
====Colony PCR====
+
-
Kil (1-16)
+
-
{|class="wikitable"
+
-
!10×Buffer||2mM dNTPs||25mM MgSO4||VF||VR||KOD Plus Neo||MilliQ||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||33||50
+
-
|}<br>
+
-
[[File:IMG_2207.jpg|200px|thumb|right]]
+
-
 
+
-
====Transformation====
+
-
*pspA-pSB1C3
+
-
*pspA-pSB3C5
+
-
====Liquid culture====
+
-
*Kil-3 →Miniprep 100.4μg/mL
+
-
*Kil-6
+
-
*Kil-7 →Miniprep 77.7μg/mL
+
-
*LacP-torA-GFP-1 →Miniprep 88.3μg/mL
+
-
*LacP-torA-GFP-2 →Miniprep 85.0μg/mL
+
-
*LacP-torA-GFP-3 →Miniprep +++
+
-
 
+
-
==August 29==
+
-
====Kil assay====
+
-
After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600.
+
-
{| class="wikitable"
+
-
|-
+
-
! scope="col"| IPTG
+
-
! scope="col"| 0
+
-
! scope="col"| 0.001mM
+
-
! scope="col"| 0.01mM
+
-
! scope="col"| 0.1mM
+
-
! scope="col"| 1mM
+
-
|-
+
-
! scope="row"| 0.5h
+
-
|0.202
+
-
|0.207
+
-
|0.201
+
-
|0.207
+
-
|0.200
+
-
|-
+
-
! scope="row"| 1h
+
-
|0.406
+
-
|0.428
+
-
|0.424
+
-
|0.402
+
-
|0.421
+
-
|-
+
-
! scope="row"| 1.5h
+
-
|0.796
+
-
|0.813
+
-
|0.779
+
-
|0.751
+
-
|0.802
+
-
|-
+
-
! scope="row"| 2h
+
-
|1.107
+
-
|1.129
+
-
|1.141
+
-
|1.092
+
-
|1.124
+
-
|-
+
-
! scope="row"| 2.5h
+
-
|1.546
+
-
|1.565
+
-
|1.541
+
-
|1.532
+
-
|1.578
+
-
|-
+
-
! scope="row"| 3h
+
-
|1.912
+
-
|1.933
+
-
|1.890
+
-
|1.883
+
-
|1.940
+
-
|-
+
-
! scope="row"| 3.5h
+
-
|2.300
+
-
|2.295
+
-
|2.238
+
-
|2.247
+
-
|2.259
+
-
|-
+
-
! scope="row"| 4h
+
-
|2.546
+
-
|2.545
+
-
|2.528
+
-
|2.490
+
-
|2.520
+
-
|-
+
-
! scope="row"| 4.5h
+
-
|2.688
+
-
|2.663
+
-
|2.603
+
-
|2.633
+
-
|2.666
+
-
|-
+
-
! scope="row"| 5h
+
-
|2.699
+
-
|2.826
+
-
|2.787
+
-
|2.593
+
-
|2.673
+
-
|-
+
-
! scope="row"| 5.5h
+
-
|2.863
+
-
|2.742
+
-
|2.754
+
-
|2.741
+
-
|2.756
+
-
|-
+
-
! scope="row"| 6h
+
-
|2.831
+
-
|2.876
+
-
|2.758
+
-
|2.759
+
-
|2.728
+
-
|-
+
-
! scope="row"| 20h
+
-
|2.671
+
-
|2.706
+
-
|2.680
+
-
|2.606
+
-
|2.619
+
-
|-
+
-
|}
+
-
 
+
-
====Liquid culture====
+
-
*pspA-pSB1C3(1-3)
+
-
*pspA-pSB3C5(1-3)
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!LacP-Kil-DT(134.7ng/μL)||EcoR1||Spe1||BufferM||MilliQ||Total
+
-
|-
+
-
|10||1||1||3||15||30
+
-
|}
+
-
37℃, overnight
+
-
====Colony PCR====
+
-
*LacP-torA-GFP-DT (1-5)
+
-
*pspA-pSB1C3 (1-5)
+
-
*pspA-pSB3C5 (1-6)
+
-
{|class="wikitable"
+
-
!10×Buffer||2mM dNTPs||25mM MgSO4||VF||VR||KOD Plus Neo||MilliQ||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||33||50
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
60℃, 30sec<br>
+
-
68℃, 40sec<br>
+
-
→30cycles<br>
+
-
====Gel extraction====
+
-
J23107-tatABCD(EcoR1, Pst1) -5.9μg/mL
+
-
[[File:0829.jpg|200px|thumb|right]]
+
-
 
+
-
==August 30==
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!J23107-tatABCD(EcoR1, Spe1)22.2μg/mL||DT(EcoR1, Xba1)37.5μg/mL||Ligation High Ver.2||Total
+
-
|-
+
-
|10||1||9||20
+
-
|}
+
-
at 16℃ for 1h
+
-
====Transformation====
+
-
J23107-tatABCD-DT (Amp resistance)
+
-
====Electrophoresis====
+
-
 
+
-
[[File:0830e.jpg|200px|thumb|right]]<br>
+
-
1. LacP-torA-GFP-DT-1<br>
+
-
2. LacP-torA-GFP-DT-2<br>
+
-
3. LacP-torA-GFP-DT-3<br>
+
-
4. LacP-torA-GFP-DT-4<br>
+
-
5. LacP-torA-GFP-DT-5<br>
+
-
6. pspA-pSB1C3-1<br>
+
-
7. pspA-pSB1C3-2<br>
+
-
8. pspA-pSB1C3-3<br>
+
-
9. pspA-pSB1C3-4<br>
+
-
10.pspA-pSB1C3-5<br>
+
-
11.pspA-pSB3C5-1<br>
+
-
12.pspA-pSB3C5-2<br>
+
-
13.pspA-pSB3C5-3<br>
+
-
14.pspA-pSB3C5-4<br>
+
-
15.pspA-pSB3C5-5<br>
+
-
16.pspA-pSB3C5-6<br>
+
-
 
+
-
====Miniprep====
+
-
*pspA-pSB3C5-1 176μg/mL
+
-
*pspA-pSB3C5-2 214μg/mL
+
-
*pspA-pSB3C5-3 461μg/mL
+
-
*pspA-pSB1C3-2 79μg/mL
+
-
====Colony PCR====
+
-
LacP-torA-GFP-DT (6-13)
+
-
{|class="wikitable"
+
-
!2×Quick Taq||VF||VR||MilliQ||Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
94℃, 2min<br>
+
-
94℃, 30sec<br>
+
-
56℃, 30sec<br>
+
-
68℃, 1min<br>
+
-
→30cycles<br><br>
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!pspA-pSB1C3||EcoR1||Spe1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|15||0.5||0.5||5||0.5||28.5||50
+
-
|}
+
-
at 37℃ for 1h
+
-
====Electrophoresis====
+
-
[[File:0830r.jpg|200px|thumb|right]]<br>
+
-
1. 1kb ladder<br>
+
-
3. pspA-pSB1C3(EcoR1, Spe1)<br>
+
-
 
+
-
==August 31==
+
-
====Gel extraction====
+
-
[[File:0831e.jpg|200px|thumb|right]]<br>
+
-
pspA-pSB1C3(EcoR1, Spe1) 8.0μg/mL
+
-
 
+
-
====Purification====
+
-
LacP-GFP-DT -0.7μg/mL
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!pspA(EcoR1, Spe1)||DT(EcoR1, Xba1)||Ligation High Ver.2||Total
+
-
|-
+
-
|25||1||13||39
+
-
|}
+
-
at 16℃ for 1h
+
-
====Transformation====
+
-
pspA-DT (Amp resistance)
+
-
 
+
-
==September 2==
+
-
====Colony PCR====
+
-
J23107-tatABCD-DT (1-8)
+
-
{|class="wikitable"
+
-
!2×Quick Taq||VF||VR||MilliQ||Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
94℃, 2min<br>
+
-
94℃, 30sec<br>
+
-
55℃, 30sec<br>
+
-
68℃, 3min<br>
+
-
→25cycles<br><br>
+
-
pspA-DT (2-8)
+
-
{|class="wikitable"
+
-
!2×Quick Taq||VF||VR||MilliQ||Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
Extension, 10sec<br>
+
-
→25cycles<br><br>
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!LacP-torA-GFP-DT||EcoR1||Spe1||BufferH||MilliQ||Total
+
-
|-
+
-
|10||1||1||2||6||20
+
-
|}
+
-
at 4℃ for overnight
+
-
====Electrophoresis====
+
-
[[File:0902.jpg|200px|thumb|right]]<br>
+
-
1. 1kb ladder<br>
+
-
3. J23107-tatABCD-DT-1<br>
+
-
4. J23107-tatABCD-DT-3<br>
+
-
5. J23107-tatABCD-DT-4<br>
+
-
6. J23107-tatABCD-DT-5<br>
+
-
7. J23107-tatABCD-DT-6<br>
+
-
8. J23107-tatABCD-DT-7<br>
+
-
9. J23107-tatABCD-DT-8<br>
+
-
[[File:0902-2.jpg|200px|thumb|right]]<br>
+
-
1. 1kb ladder<br>
+
-
3. J23107-tatABCD-DT-1<br>
+
-
4. J23107-tatABCD-DT-3<br>
+
-
5. J23107-tatABCD-DT-4<br>
+
-
6. J23107-tatABCD-DT-5<br>
+
-
7. J23107-tatABCD-DT-6<br>
+
-
8. J23107-tatABCD-DT-7<br>
+
-
9. J23107-tatABCD-DT-8<br>
+
-
11. LacP-pSB3C5(Spe1, Pst1)
+
-
[[File:0902-3.jpg|200px|thumb|right]]<br>
+
-
1. 1kb ladder<br>
+
-
3. pspA-DT-2<br>
+
-
4. pspA-DT-3<br>
+
-
5. pspA-DT-4<br>
+
-
6. pspA-DT-5<br>
+
-
7. pspA-DT-6<br>
+
-
8. pspA-DT-7<br>
+
-
9. pspA-DT-8<br>
+
-
 
+
-
==September 3==
+
-
====PCR====
+
-
tatABCD-DT
+
-
{|class="wikitable"
+
-
!10×Buffer||2mM dNTPs||25mM MgSO4||VF||VR||KOD Plus Neo||MilliQ||DNA||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||32||1||50
+
-
|}
+
-
====Gel extraction====
+
-
[[File:0903.jpg|200px|thumb|right]]
+
-
LacP-torA-GFP-DT →See "September 5 -Gel extraction-"<br><br><br><br><br>
+
-
 
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!torA-GFP-DT||Xba1||Pst1||BufferM||MilliQ||Total
+
-
|-
+
-
|20||2||2||4||12||40
+
-
|}
+
-
====Purification====
+
-
J23107-tatABCD 29.6μg/mL 1.56(260/280) 1.60(260/230)
+
-
====Electrophoresis====
+
-
[[File:0903-2.jpg|200px|thumb|right]]
+
-
1. 1kb ladder<br>
+
-
2. J23107-tatABCD<br><br><br><br><br>
+
-
 
+
-
====Liquid culture====
+
-
*LacP-kil-DT(1)
+
-
*pspA-DT(4, 7)
+
-
====PCR====
+
-
J23107-tatABCD
+
-
{|class="wikitable"
+
-
!10×Buffer||2mM dNTPs||25mM MgSO4||VF||VR||KOD Plus Neo||MilliQ||DNA||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||29||4||50
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
60℃, 30sec<br>
+
-
68℃, 10sec<br>
+
-
→30cycles<br><br>
+
-
====Gel extraction====
+
-
[[File:IMG_2135.jpg|200px|thumb|right]]
+
-
LacP-torA-GFP-DT 31μg/mL 1.28(260/280) 0.49(260/230)<br>
+
-
LacP-torA-GFP-DT 8μg/mL 1.32(260/280) 0.83(260/230)<br><br><br><br><br>
+
-
 
+
-
====Transformation====
+
-
J23107-tatABCD (Amp resistance)<br>
+
-
J23113-kil-DT (Amp resistance)<br>
+
-
J23100-kil-DT (CP resistance)<br>
+
-
J23101-kil-DT (CP resistance)<br>
+
-
J23106-kil-DT (CP resistance)<br>
+
-
J23115-kil-DT (CP resistance)→We didn't transform because of lack of culture medium.<br>
+
-
J23105-kil-DT (CP resistance)→We didn't transform because of lack of culture medium.<br>
+
-
====Electrophoresis====
+
-
[[File:IMG_2136.jpg|200px|thumb|right]]
+
-
1. 1kb ladder<br>
+
-
2-7. LacP-torA-GFP-DT<br><br><br><br>
+
-
 
+
-
====Colony PCR====
+
-
LacP-torA-GFP-DT (14-21)
+
-
J23113-kil-DT (Amp resistance, 1-4)
+
-
J23113-kil-DT (CP resistance, 1-4)
+
-
{|class="wikitable"
+
-
!2×Quick Taq||primer-f||primer-r||MilliQ||Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
94℃, 2min<br>
+
-
94℃, 30sec<br>
+
-
55℃, 30sec<br>
+
-
68℃, 1min<br>
+
-
→25cycles<br><br>
+
-
 
+
-
==September 4==
+
-
====Liquid culture====
+
-
*LacP-kil-DT(4, 5)(Amp resistance)
+
-
*pspA-DT(3, 6, 7)(Amp resistance)
+
-
====PCR====
+
-
We did PCR 10 more cycles to amplify products of PCR that we had done yesterday because we could not amplify J23107-tatABCD on September 3.<br>
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
60℃, 30sec<br>
+
-
68℃, 1.5min<br>
+
-
→30cycles<br><br>
+
-
====Electrophoresis====
+
-
[[File:IMG_2137.jpg|200px]]<br>
+
-
1-8. LacP-torA-GFP-DT<br><br>
+
-
[[File:IMG_2138.jpg|200px]]<br>
+
-
1-4. J23113-kil-DT (Amp resistance)<br>
+
-
5-8. J23113-kil-DT (CP resistance)<br>
+
-
 
+
-
====Liquid culture====
+
-
*LacP-torA-GFP-DT
+
-
*J23113-kil-DT
+
-
*LacP-kil-DT(with IPTG)
+
-
*LacP-kil-DT(without IPTG)
+
-
After 20 hour, we quantified OD600.
+
-
{|class="wikitable"
+
-
!plasmid||OD600
+
-
|-
+
-
|LacP-kil-DT(with IPTG)||2.116
+
-
|-
+
-
|LacP-kil-DT(without IPTG)||2.320
+
-
|}
+
-
We cultured CP tolerance plasmid and give an antibiotic(Amp or Kan or none) after 2 hours.
+
-
We quantify OD600.
+
-
{|class="wikitable"
+
-
!time||1||2||3
+
-
|-
+
-
|2hours||0.131||0.100||0.606
+
-
|-
+
-
|add an antibiotic||Amp||Kan||none
+
-
|-
+
-
|7hours||0.491||0.988||2.634
+
-
|-
+
-
|19hours||2.253||0.815||2.742
+
-
|}
+
-
 
+
-
==September 5==
+
-
====Colony PCR====
+
-
J23107-tatABCD (1-7)
+
-
{|class="wikitable"
+
-
!buffer||2mM dNTPs||25mM MgSO4||primer-f||primer-r||KOD Plus Neo||DW||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||33||50
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
60℃, 30sec<br>
+
-
68℃, 1.25min<br>
+
-
→30cycles<br><br>
+
-
====Gel extraction====
+
-
<small>by Kato</small><br>
+
-
See "September 3 -Gel extraction-"
+
-
LacP-torA-GFP-DT 15.9μg/mL 1.29(260/280) 0.38(260/230)
+
-
LacP-torA-GFP-DT 17.2μg/mL 1.23(260/280) 0.48(260/230)
+
-
====Miniprep====
+
-
*J23113-kil-DT 85.7μg/mL 1.76(260/280) 1.97(260/230)
+
-
*LacP-torA-GFP-DT 126.6μg/mL 1.81(260/280) 2.09(260/230)
+
-
*LacP-kil-DT-4 62.5μg/mL 1.79(260/280) 1.99(260/230)
+
-
*LacP-kil-DT-5 56.0μg/mL 1.79(260/280) 2.01(260/230)
+
-
*pspA-DT-3 322μg/mL 1.45(260/280) 1.42(260/230)
+
-
*pspA-DT-6 147.6μg/mL 1.25(260/280) 1.37(260/230)
+
-
*pspA-DT-7 145.5μg/mL 1.24(260/280) 1.27(260/230)
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!pspA-DT-6||Xba1||Pst1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|20||0.5||0.5||3||0.5||5.5||30
+
-
|}
+
-
at 37℃ for 1h
+
-
====Electrophoresis====
+
-
[[File:IMG_2139.jpg|200px|thumb|right]]
+
-
J23107-tatABCD (1-7)<br><br><br><br><br><br><br><br>
+
-
 
+
-
====Colony PCR====
+
-
J23107-tatABCD (8-23)
+
-
{|class="wikitable"
+
-
!2×Quick Taq||VF2||VR||MilliQ||Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
94℃, 2min<br>
+
-
94℃, 30sec<br>
+
-
55℃, 30sec<br>
+
-
68℃, 1.5min<br>
+
-
→30cycles<br><br>
+
-
====Gel extraction====
+
-
pspA-DT 10.2μg/mL 1.42(260/280) 0.76(260/230)
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!pspA-DT(Xba1, Pst1)||LacP(pSB3C5)(Spe1, Pst1)||Ligation High Ver.2||Total
+
-
|-
+
-
|5||1||3||9
+
-
|}
+
-
at 16℃
+
-
====Electrophoresis====
+
-
[[File:IMG_2140.jpg|200px]]<br>
+
-
LacP-torA-GFP-DT(8-15)<br><br>
+
-
[[File:IMG_2141.jpg|200px]]<br>
+
-
LacP-torA-GFP-DT(9-23)<br>
+
-
 
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!LacP-torA-GFP-DT(EcoR1, Spe1)||DT 17.2μg/mL(Xba1, Pst1)||Ligation High Ver.2||Total
+
-
|-
+
-
|15||25||20||60
+
-
|}
+
-
====Liquid culture====
+
-
*J23107-tatABCD-8
+
-
*J23107-tatABCD-20
+
-
*LacP-torA-GFP-DT-21
+
-
 
+
-
==September 6==
+
-
====Miniprep====
+
-
*J23107-tatABCD-8 82.4μg/mL 2.06(260/280) 2.95(260/230)
+
-
*J23107-tatABCD-20 102.9μg/mL 2.04(260/280) 2.94(260/230)
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!J23107-tatABCD 102.9μg/mL||Spe1||Pst1||BufferH||BSA||MilliQ||Total
+
-
|-
+
-
|10||0.5||0.5||3||0.5||15.5||30
+
-
|}
+
-
at 37℃ for 1h
+
-
====Transformation====
+
-
LacP-pspA-DT(pSB3C5)
+
-
{|class="wikitable"
+
-
!DNA||Competent cell||Total
+
-
|-
+
-
|2||20||22
+
-
|}
+
-
====Gel extraction====
+
-
[[File:IMG_2142.jpg|200px|thumb|right]]<br>
+
-
J23107-tatABCD(Spe1, Pst1) <br>
+
-
→We failed gel extraction because we mistook steps of experiment.<br><br><br><br><br><br>
+
-
 
+
-
====Electrophoresis====
+
-
[[File:IMG_2143.jpg|200px|thumb|right]]<br><br>
+
-
LacP-torA-GFP-DT-pspA-DT<br><br><br><br><br>
+
-
 
+
-
====Restrictive Digestion====
+
-
<small>by Terasaka</small><br>
+
-
{| class="wikitable"
+
-
!J23107-tatABCD||Spe1||Pst1||BufferH||BSA||MilliQ||Total
+
-
|-
+
-
|10||0.5||0.5||3||0.5||15.5||30
+
-
|}
+
-
====Gel extraction====
+
-
[[File:IMG_2144.jpg|200px|thumb|right]]<br>
+
-
J23107-tatABCD(Spe1, Pst1) <br>
+
-
→See "September 7 -Gel extraction-"<br><br><br><br><br>
+
-
 
+
-
==September 7==
+
-
====Liquid culture====
+
-
*LacP-torA-GFP-DT-1 (CP resistance)
+
-
*LacP-torA-GFP-DT-21 (CP resistance)
+
-
preculture for 1 hour
+
-
====Colony PCR====
+
-
LacP-torA-GFP-DT (1-8)
+
-
LacP-pspA-DT (1-8)
+
-
{|class="wikitable"
+
-
!2×Quick Taq||VF2||VR||MilliQ||Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
94℃, 2min<br>
+
-
94℃, 30sec<br>
+
-
55℃, 30sec<br>
+
-
68℃, 1.5min<br>
+
-
→30cycles<br><br>
+
-
====Restrictive Digestion====
+
-
<small>by Terasaka</small><br>
+
-
{| class="wikitable"
+
-
!J23107-tatABCD||EcoR1||Spe1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|10||0.5||0.5||3||0.5||15.5||30
+
-
|}
+
-
{| class="wikitable"
+
-
!GFP-DT||Xba1||Pst1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|15||0.5||0.5||3||0.5||10.5||30
+
-
|}
+
-
====Electrophoresis====
+
-
[[File:IMG_2145.jpg|200px|thumb|right]]<br>
+
-
① -⑧. LacP-pspA-DT<br>
+
-
1-8. LacP-torA-GFP-DT<br><br><br><br><br>
+
-
====Gel extraction====
+
-
[[File:IMG_2146.jpg|200px|thumb|right]]<br>
+
-
*J23107-tatABCD(EcoR1, Spe1)
+
-
*J23107-tatABCD(Pst1, Spe1) ①57.6μg/mL ②23.4μg/mL
+
-
*GFP-DT(Xba1, Pst1) <br><br><br><br>
+
-
 
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!pspA-DT(145.5μg/mL)||Xba1||Pst1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|20||0.5||0.5||3||0.5||5.5||30
+
-
|}
+
-
37℃, overnight
+
-
====Liquid culture====
+
-
*LacP-torA-GFP-DT-7 (LB 2mL)<br>
+
-
+IPTG 0mM, 0.05mM, 0.1mM, 0.15mM, 0.2mM, 0.3mM
+
-
*LacP-torA-GFP-DT-7 (LB 5mL)
+
-
 
+
-
==September 8==
+
-
====Check the effect of Amp====
+
-
We used 5mL of the liquid culture medium with LacP-torA-GFP-DT-7.<br>
+
-
→See "September 7 -Liquid culture-"<br>
+
-
We diluted it with LB until OD600=0.444, and dispensed it.<br>
+
-
The dispense volume was 1.3mL.<br>
+
-
We added 0/13/130μL of Amp(50mg/mL) to it.<br>
+
-
=====5.5h after incubating at 37℃=====
+
-
{| class="wikitable"
+
-
!Amp||0μL||13μL||130μL
+
-
|-
+
-
|OD600||2.457||2.696||0.310
+
-
|}
+
-
====Observation through a confocal microscope====
+
-
We used the liquid culture media with LacP-torA-GFP-DT + 13μL of Amp(50mg/mL) + IPTG 0/0.1/0.15mM.<br>
+
-
Their OD600 was 0.47.<br>
+
-
3h after incubating at 37℃, we eliminated each supernatant using a centrifuge, and diluted them with LB to which Amp was added.<br>
+
-
2.5h after incubating at 37℃, we observed them through a confocal microscope.<br>
+
-
We failed to observe it.
+
-
====Gel extraction====
+
-
[[File:IMG_2147.jpg|200px|thumb|right]]
+
-
pspA-DT -5.5μg/mL<br><br><br>
+
-
 
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!pspA-DT-3(322μg/mL)||Xba1||Pst1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|10||1||1||3||3||12||30
+
-
|}
+
-
at 37℃ for 1h
+
-
 
+
-
==September 9==
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!J23107-tatABCD(102.9μg/mL)||EcoR1||Spe1||BufferM||MilliQ||Total
+
-
|-
+
-
|10||1||1||3||15||30
+
-
|}
+
-
====Electrophoresis====
+
-
[[File:IMG_2148.jpg|200px|thumb|right]]<br>
+
-
1. J23107-tatABCD(EcoR1, Spe1)<br>
+
-
2. J23107-tatABCD(before restriction)<br>
+
-
3. pspA-DT(Xba1, Pst1)<br>
+
-
4. pspA-DT(before restriction)<br><br><br>
+
-
 
+
-
====Gel extraction====
+
-
[[File:IMG_2149.jpg|200px|thumb|right]]<br>
+
-
*J23107-tatABCD(EcoR1, Spe1) 26μg/mL
+
-
*pspA-DT(Xba1, Pst1) 199μg/mL
+
-
<br><br><br>
+
-
 
+
-
====Liquid culture====
+
-
*J23100/J23101/J23106-1
+
-
*pspA-DT-5 ×2
+
-
*LacP-torA-GFP-DT-21
+
-
*GFP generator-2
+
-
*LacP-pspA-DT-6
+
-
 
+
-
==September 10==
+
-
====Miniprep====
+
-
*J23100-1  105.5μg/mL
+
-
*J23101-1  76.0μg/mL
+
-
*J23106-1  90.9μg/mL
+
-
*pspA-DT-5  ①104.1μg/mL  ②80.0μg/mL
+
-
*LacP-torA-GFP-DT-21  79.6μg/mL
+
-
*GFP generator-2  84.1μg/mL
+
-
*LacP-pspA-DT-6  99.8μg/mL
+
-
====Ligation====
+
-
*J23107-tatABCD-DT
+
-
{| class="wikitable"
+
-
!J23107-tatABCD(EcoR1, Spe1)||DT(EcoR1, Xba1)||Ligation High Ver.2||Total
+
-
|-
+
-
|10||5||7.5||22.5
+
-
|}
+
-
*Negative control for J23107-tatABCD-DT
+
-
{| class="wikitable"
+
-
!DT(EcoR1, Xba1)||Ligation High Ver.2||Total
+
-
|-
+
-
|5||5||10
+
-
|}
+
-
*J23107-tatABCD-pspA-DT
+
-
{| class="wikitable"
+
-
!J23107-tatABCD(Spe1, Pst1)||pspA-DT(Xba1, Pst1)||Ligation High Ver.2||Total
+
-
|-
+
-
|3||6||9||18
+
-
|}
+
-
*Negative control for J23107-tatABCD-pspA-DT
+
-
{| class="wikitable"
+
-
!J23107-tatABCD(Spe1, Pst1)||Ligation High Ver.2||Total
+
-
|-
+
-
|3||3||6
+
-
|}
+
-
====Restrictive Digestion====
+
-
[[File:IMG_2151.jpg|200px|thumb|right]]
+
-
{| class="wikitable"
+
-
!DNA||Spe1||Pst1||BufferH||MilliQ||Total
+
-
|-
+
-
|15||1||1||3||10||30
+
-
|}
+
-
DNA=J23100(105.5μg/mL), J23101(76.0μg/mL), J23106(90.9μg/mL)<br>
+
-
at 37℃<br><br>
+
-
====Gel extraction====
+
-
[[File:IMG_2152.jpg|200px]]
+
-
[[File:IMG_2153.jpg|200px]]<br>
+
-
J23100  27.1μg/mL<br>
+
-
J23101  50.2μg/mL<br>
+
-
J23106  16.1μg/mL<br>
+
-
====Ligation====
+
-
*J23100/J23101/J23106-kil-DT
+
-
{| class="wikitable"
+
-
!J23100/J23101/J23106(Spe1, Pst1)||kil-DT(Xba1, Pst1)||Ligation High Ver.2||Total
+
-
|-
+
-
|1||5||3||9
+
-
|}
+
-
*Negative control for J23100/J23101/J23106-kil-DT
+
-
{| class="wikitable"
+
-
!J23100/J23101/J23106(Spe1, Pst1)||MilliQ||Ligation High Ver.2||Total
+
-
|-
+
-
|1||5||3||9
+
-
|}
+
-
====Transformation====
+
-
J23100/J23101/J23106-kil-DT
+
-
 
+
-
==September 11==
+
-
====Colony PCR====
+
-
*J23107-tatABCD-DT (1-8)
+
-
*J23107-tatABCD-pspA-DT (1-8)
+
-
{|class="wikitable"
+
-
!2×Quick Taq||VF2||VR||MilliQ||Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
94℃, 2min<br>
+
-
94℃, 30sec<br>
+
-
55℃, 30sec<br>
+
-
68℃, 3.5min<br>
+
-
→30cycles
+
-
====Electrophoresis====
+
-
*J23107-tatABCD-DT (1-8) 
+
-
[[File:IMG_2156.jpg|200px]]
+
-
*J23107-tatABCD-pspA-DT (1-8)
+
-
[[File:IMG_2155.jpg|200px]]
+
-
 
+
-
====Restrictive Digestion====
+
-
[[File:IMG_2154.jpg|200px|thumb|right]]
+
-
{| class="wikitable"
+
-
!GFP generator(84.1μg/mL)||Xba1||Pst1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|15||1||1||3||3||7||30
+
-
|}
+
-
{| class="wikitable"
+
-
!LacP-torA-GFP-DT(85.0μg/mL)||Spe1||Pst1||BufferH||MilliQ||Total
+
-
|-
+
-
|15||1||1||3||10||30
+
-
|}
+
-
{| class="wikitable"
+
-
!LacP-pspA-DT(99.8μg/mL)||Xba1||Pst1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|15||1||1||3||3||7||30
+
-
|}
+
-
 
+
-
====Liquid culture====
+
-
pSB1C3(self-ligation) ×5 in LB 3mLCP 3μL<br>
+
-
When OD600=approximately 0.6, we added 0/30/60/150/300μL of Amp(50mg/mL) to each liquid culture medium.<br>
+
-
1h and 5h after incubating at 37℃, we measured OD600.<br>
+
-
{| class="wikitable"
+
-
!||1h||5h
+
-
|-
+
-
|0||-0.020||0.403
+
-
|-
+
-
|30||-0.046||0.544
+
-
|-
+
-
|60||0.263||0.007
+
-
|-
+
-
|150||0.061||0.839
+
-
|-
+
-
|300||0.093||1.441
+
-
|}
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!pSB3C5(EcoR1, Pst1)||pspA-DT(Xba1, Pst1)||J23107-tatABCD(EcoR1, Spe1)||Ligation High Ver.2||Total
+
-
|-
+
-
|1||3||5||4.5||13.5
+
-
|}
+
-
4℃, overnight
+
-
====Colony PCR====
+
-
*J23100-kil-DT (1-6)
+
-
*J23101-kil-DT (1-7)
+
-
*J23106-kil-DT (1-2)
+
-
{|class="wikitable"
+
-
!2×Quick Taq||VF||VR||MilliQ||Total
+
-
|-
+
-
|23||1||1||25||50
+
-
|}
+
-
====Gel extraction====
+
-
[[File:IMG_2159.jpg|200px|thumb|right]]<br>
+
-
*GFP generator (Xba1, Pst1) 14.1μg/mL
+
-
*LacP-torA-GFP-DT (Spe1, Pst1) 42.9μg/mL 1.13(260/280) 1.19(260/230)
+
-
*LacP-pspA-DT (Xba1, Pst1) 20.0μg/mL 1.01(260/280) 1.30(260/230)
+
-
<br><br>
+
-
====Electrophoresis====
+
-
[[File:IMG_2160.jpg|200px|thumb|right]]<br>
+
-
*J23107-tatABCD-DT (1-8)
+
-
*J23106-kil-DT (1, 2)
+
-
*J23100-kil-DT (1-4)
+
-
<br><br>
+
-
====Liquid culture====
+
-
*LacP-torA-GFP-DT
+
-
*pSB1C3
+
-
====Colony PCR====
+
-
We did PCR 10 more cycle to amplify products of PCR on 9/11 of J23107-tatABCD-pspA-DT and J23107-tatABCD-DT.
+
-
<br>
+
-
==September 12==
+
-
====Colony PCR====
+
-
J23107-tatABCD-pspA-DT (9-15)
+
-
94℃, 2min<br>
+
-
94℃, 30sec<br>
+
-
55℃, 30sec<br>
+
-
68℃, 4min<br>
+
-
→26cycles<br><br>
+
-
====Check the effect of Amp====
+
-
We cultured pSB1C3 for overnight.<br>
+
-
→See "September 7 -Liquid culture-"<br>
+
-
We diluted it with LB, and dispensed it.<br>
+
-
We added Amp(50mg/mL) to it until density of Amp was 1/10, 1/30, 1/50 .<br>
+
-
{|class="wikitable"
+
-
!Amp||start||5 hours
+
-
|-
+
-
|1/10||0.557||0.077
+
-
|-
+
-
|1/30||0.628||0.166
+
-
|-
+
-
|1/50||0.472||0.055
+
-
|-
+
-
|0||0.594||2.585
+
-
|}
+
-
====Electrophoresis====
+
-
[[File:IMG_2162.jpg|200px]]
+
-
*J23107-tatABCD-pspA-DT
+
-
[[File:IMG_2161.jpg|200px]]
+
-
*J23101-kil-DT (1-7)
+
-
 
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!pspA(pSB3C5) (106μg/mL)||EcoR1||Pst1||BufferH||MilliQ||Total
+
-
|-
+
-
|15||1||1||3||10||30
+
-
|}
+
-
{| class="wikitable"
+
-
!pspA-DT-3||Xba1||Pst1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|15||1||1||3||3||7||30
+
-
|}
+
-
{| class="wikitable"
+
-
!J23107-tatABCD (102.4μg/mL)||EcoR1||Spe1||BufferM||MilliQ||Total
+
-
|-
+
-
|15||1||1||3||10||30
+
-
|}
+
-
====Electrophoresis====
+
-
[[File:IMG_2165.jpg|200px|thumb|right]]
+
-
*pspA(pSB3C5)(EcoR1, Pst1)
+
-
*pspA-DT(Xba1, Pst1)
+
-
*J23107-tatABCD(EcoR1, Spe1)
+
-
*pSB1C3(EcoR1, Spe1)
+
-
<br><br><br>
+
-
 
+
-
====Transformation====
+
-
J23107-tatABCD-pspA-DT(pSB3C5)
+
-
{|class="wikitable"
+
-
!DNA||Competent cell||Total
+
-
|-
+
-
|2||20||22
+
-
|}
+
-
on ice for 30 minites
+
-
heatshock at 42℃ for 1 hour
+
-
on ice 2 minites
+
-
then add SOC medium 200μl
+
-
preculture at 37℃ for 1 hour
+
-
incubate CP culture plate
+
-
====Gel extraction====
+
-
[[File:IMG_2166.jpg|200px|thumb|right]]
+
-
*pspA(pSB3C5)(EcoR1, Pst1) 26.8μg/mL 1.27(260/280) 0.32(260/230)
+
-
*pspA-DT(Xba1, Pst1) 26.1μg/mL 1.13(260/280) 0.55(260/230)
+
-
*J23107-tatABCD(EcoR1, Spe1) 1.5μg/mL 1.99(260/280) 0.06(260/230)
+
-
<br><br><br>
+
-
 
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!pSB3C5(EcoR1, Pst1) 26.8μg/mL||pspA-DT(Xba1, Pst1) 26.1μg/mL||J23107-tatABCD(EcoR1, Spe1) 1.5μg/mL||Ligation High Ver.2||Total
+
-
|-
+
-
|1||3||6||5||15
+
-
|}
+
-
{| class="wikitable"
+
-
!pSB3C5(EcoR1, Pst1) 26.8μg/mL||MilliQ||Ligation High Ver.2||Total
+
-
|-
+
-
|1||9||5||15
+
-
|}
+
-
{| class="wikitable"
+
-
!LacP(pSB3C5)(Spe1, Pst1) 10μg/mL||GFP-DT(Xba1, Pst1) 7.0μg/mL||Ligation High Ver.2||Total
+
-
|-
+
-
|1||5||3||9
+
-
|}
+
-
{| class="wikitable"
+
-
!LacP(pSB3C5)(Spe1, Pst1) 10μg/mL||MilliQ||Ligation High Ver.2||Total
+
-
|-
+
-
|1||5||3||9
+
-
|}
+
-
4℃, overnight
+
-
====Observation through a confocal microscope====
+
-
left:observation with Hoechst<br>
+
-
right:observation GFP<br>
+
-
[[File:F17822ea-b51e-49f5-aa44-8dc8638a1ff6.png|200px]]
+
-
[[File:E01af7d0-908e-4fcc-9eb3-5b92aa802224.png|200px]]
+
-
 
+
-
==September 13==
+
-
 
+
-
+
-
 
+
-
+
-
 
+
-
 
+
-
 
+
-
 
+
-
====Miniprep====
+
-
 
+
-
+
-
 
+
-
+
-
 
+
-
 
+
-
 
+
-
 
+
-
*J23107-tatABCD-pspA-DT(pSB3C5)-1 130μg/mL 1.37(260/280) 0.79(260/230)
+
-
 
+
-
 
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!J23107-tatABCD-pspA-DT(pSB3C5) (130μg/mL)||Spe1||Pst1||BufferH||MilliQ||Total
+
-
 
+
-
 
+
-
|-
+
-
|10||1||1||3||15||30
+
-
 
+
-
|}
+
-
====Transformation====
+
-
 
+
-
*LacP-GFP generater
+
-
 
+
-
*LacP
+
-
*J23107-tatABCD-pspA-DT(pSB3C5)
+
-
*J23107-tatABCD
+
-
====Colony PCR====
+
-
 
+
-
J23107-tatABCD-pspA-DT<br>
+
-
 
+
-
→See "September 14 -Colony PCR-"
+
-
 
+
-
==September 14==
+
-
====Colony PCR====
+
-
[[File:IMG_2169.jpg|200px|thumb|right]]
+
-
See "September 13 -Colony PCR-"<br>
+
-
J23107-tatABCD-pspA-DT<br>
+
-
<br><br><br><br><br><br>
+
-
 
+
-
====Colony PCR====
+
-
*J23107-tatABCD-pspA-DT (1-6)
+
-
{|class="wikitable"
+
-
!2×Quick Taq||VF||VR||MilliQ||Total
+
-
|-
+
-
|23||1||1||25||50
+
-
|}
+
-
94℃, 2min<br>
+
-
94℃, 30sec<br>
+
-
55℃, 30sec<br>
+
-
68℃, 4min<br>
+
-
→30cycles<br><br>
+
-
 
+
-
==September 15==
+
-
====Liquid culture====
+
-
*J23107-tatABCD-pspA-DT-1
+
-
*J23107-tatABCD-pspA-DT-2
+
-
*J23107-tatABCD-pspA-DT-3
+
-
====Miniprep====
+
-
*J23107-tatABCD-pspA-DT-1 22.9μg/mL
+
-
*J23107-tatABCD-pspA-DT-2 51.8μg/mL 1.57(260/280) 1.01(260/230)
+
-
*J23107-tatABCD-pspA-DT-3 28.5μg/mL 1.57(260/280) 0.94(260/230)
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!J23107-tatABCD-pspA-DT (51.8μg/mL)||EcoR1||Spe1||BufferM||MilliQ||Total
+
-
|-
+
-
|15||0.5||0.5||3||11||30
+
-
|}
+
-
{| class="wikitable"
+
-
!LacP-torA-GFP-DT (79.6μg/mL)||EcoR1||Xba1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|10||0.5||0.5||3||0.5||15.5||30
+
-
|}
+
-
{| class="wikitable"
+
-
!LacP-kil-DT (56.0μg/mL)||EcoR1||Xba1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|15||0.5||0.5||3||0.5||10.5||30
+
-
|}
+
-
{| class="wikitable"
+
-
!J23107-tatABCD-pspA-DT (51.8μg/mL)||EcoR1||Pst1||BufferM||MilliQ||Total
+
-
|-
+
-
|15||0.5||0.5||3||11||30
+
-
|}
+
-
37℃, 2 hours
+
-
====Electrophoresis====
+
-
[[File:IMG_2170.jpg|200px|thumb|right]]
+
-
①. 1kb ladder<br>
+
-
②. J23107-tatABCD-pspA-DT<br>
+
-
③. J23107-tatABCD-pspA-DT(EcoR1, Spe1)<br>
+
-
④. J23107-tatABCD-pspA-DT(EcoR1, Pst1)<br>
+
-
⑤. LacP-kil-DT<br>
+
-
⑥. LacP-kil-DT(EcoR1, Xba1)<br>
+
-
⑦. LacP-torA-GFP-DT<br>
+
-
⑧. LacP-torA-GFP-DT(EcoR1, Xba1)<br>
+
-
 
+
-
====Gel extraction====
+
-
[[File:IMG_2171.jpg|200px]]
+
-
[[File:IMG_2172.jpg|200px]]<br>
+
-
①. 1kb ladder<br>
+
-
②. J23107-tatABCD-pspA-DT(EcoR1, Spe1)<br>
+
-
③. J23107-tatABCD-pspA-DT(EcoR1, Spe1)<br>
+
-
④. J23107-tatABCD-pspA-DT(EcoR1, Pst1)<br>
+
-
⑤. J23107-tatABCD-pspA-DT(EcoR1, Pst1)<br>
+
-
⑥. 1kb ladder<br>
+
-
*J23107-tatABCD-pspA-DT(EcoR1, Spe1) 14.2μg/mL 1.28(260/280) 0.54(260/230)
+
-
*J23107-tatABCD-pspA-DT(EcoR1, Pst1) 7.3μg/mL 1.55(260/280) 0.38(260/230)
+
-
 
+
-
====Purification====
+
-
*LacP-torA-GFP-DT(EcoR1, Xba1) 7.7μg/mL 1.41(260/280) 0.99(260/230)
+
-
*LacP-kil-DT(EcoR1, Xba1) →failed
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!LacP-torA-GFP-DT(EcoR1, Xba1)||J23107-tatABCD-pspA-DT(EcoR1, Spe1)||Ligation High Ver.2||Total
+
-
|-
+
-
|4||10||7||21
+
-
|}
+
-
{| class="wikitable"
+
-
!pSB1C3(EcoR1, Pst1)||J23107-tatABCD-pspA-DT(EcoR1, Pst1)||Ligation High Ver.2||Total
+
-
|-
+
-
|4||10||7||21
+
-
|}
+
-
at 16℃ for overnight
+
-
 
+
-
==September 16==
+
-
====Transformation====
+
-
LacP-torA-GFP-DT-J23107-tatABCD-pspA-DT<br>
+
-
J23107-tatABCD-pspA-DT(pSB1C3)<br>
+
-
==September 17==
+
-
====Colony PCR====
+
-
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT (1-6)<br>
+
-
J23107-tatABCD-pspA-DT(pSB1C3) (1-6)<br>
+
-
J23107-kil-DT (6-10)
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!LacP-torA-GFP-DT (126μg/mL)||EcoR1||Xba1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|10||1||1||3||3||12||30
+
-
|}
+
-
{| class="wikitable"
+
-
!LacP-torA-GFP-DT (126μg/mL)||Xba1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|3||1||3||3||20||30
+
-
|}
+
-
====Electrophoresis====
+
-
[[File:IMG_2173.jpg|200px|thumb|right]]
+
-
A1-A6. J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT (1-6)<br>
+
-
B1-B6. J23107-tatABCD-pspA-DT(pSB1C3) (1-6)<br><br><br><br><br><br><br>
+
-
 
+
-
====Liquid culture====
+
-
*J23107-tatABCD-pspA-DT(pSB1C3)
+
-
*J23107-tatABCD-pspA-DT(pSB3C5)
+
-
*LacP-torA-GFP-DT(pSB3C5)
+
-
====Electrophoresis====
+
-
[[File:IMG_2174.jpg|200px|thumb|right]]
+
-
lane1. 1kb ladder<br>
+
-
lane2. J23101-kil-DT-6<br>
+
-
lane3. J23101-kil-DT-7<br>
+
-
lane4. J23101-kil-DT-8<br>
+
-
lane5. J23101-kil-DT-10<br>
+
-
lane6. LacP-torA-GFP-DT<br>
+
-
lane7. LacP-torA-GFP-DT(EcoR1, Xba1)<br>
+
-
lane8. LacP-torA-GFP-DT(Xba1)<br>
+
-
 
+
-
====Miniprep====
+
-
*J23106-kil-DT 11.7μg/mL 1.93(260/280) 1.46(260/230)
+
-
 
+
-
==September 18==
+
-
====Gel extraction====
+
-
*LacP-torA-GFP-DT(EcoR1, Xba1) 36.9μg/mL 1.37(260/280) 0.82(260/230)
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!J23107-tatABCD-pspA-DT(EcoR1, Spe1) 14.2μg/mL||LacP-torA-GFP-DT(EcoR1, Xba1) 36.9μg/mL||Ligation High Ver.2||Total
+
-
|-
+
-
|8||1.5||9.5||19
+
-
|}
+
-
{| class="wikitable"
+
-
!LacP-torA-GFP-DT(EcoR1, Xba1) 36.9μg/mL||Ligation High Ver.2||Total
+
-
|-
+
-
|1.5||1.5||3
+
-
|}
+
-
at 4℃ for 1 hour
+
-
====Transformation====
+
-
*J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT
+
-
*LacP-torA-GFP-DT(EcoR1, Xba1)
+
-
====Observation through a confocal microscope====
+
-
We made sample for observation through a confocal microscope.<br>
+
-
We incubated sample with 0mM/0.5mM IPTG for 1.5 hours.<br>
+
-
Then, we incubated onto medium without IPTG for 2.5 hours.<br>
+
-
 
+
-
{| class="wikitable"
+
-
!IPTG(mM)||medium
+
-
|-
+
-
|0||LB
+
-
|-
+
-
|0.1||LB
+
-
|-
+
-
|0.1||LB with 0.1mM IPTG  (positive control)
+
-
|-
+
-
|0||LB with 2 percent glucose
+
-
|-
+
-
|0.1||LB with 2 percent glucose
+
-
|-
+
-
|0||LB with 5 percent glucose
+
-
|-
+
-
|0.1||LB with 5 percent glucose
+
-
|-
+
-
|0||LB with 2 percent glucose (make medium new after incubate for 1.5 hours)
+
-
|-
+
-
|0||LB with 2 percent glucose (make medium new after incubate for 1.5 hours)
+
-
|}
+
-
====Restrictive Digestion====
+
-
{| class="wikitable"
+
-
!pSB4K5||EcoR1||Pst1||BufferH||MilliQ||Total
+
-
|-
+
-
|10||1||1||3||15||30
+
-
|}
+
-
{| class="wikitable"
+
-
!pSB4K5||EcoR1||BufferH||MilliQ||Total
+
-
|-
+
-
|3||0.5||1||5.5||30
+
-
|}
+
-
{| class="wikitable"
+
-
!pSB4K5||Pst1||BufferH||MilliQ||Total
+
-
|-
+
-
|3||0.5||1||5.5||10
+
-
|}
+
-
{| class="wikitable"
+
-
!LacP-torA-GFP-DT||Xba1||Pst1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|10||1||1||3||3||12||30
+
-
|}
+
-
{| class="wikitable"
+
-
!LacP-torA-GFP-DT||Xba1||BufferM||BSA||MilliQ||Total
+
-
|-
+
-
|2||1||1||1||5||10
+
-
|}
+
-
{| class="wikitable"
+
-
!LacP-torA-GFP-DT||Pst1||BufferH||MilliQ||Total
+
-
|-
+
-
|2||1||1||6||10
+
-
|}
+
-
37℃, 2 hours
+
-
====Gel extraction====
+
-
*LacP-torA-GFP-DT(Xba1, Pst1) 9.7μg/mL 1.34(260/280) 0.62(260/230)
+
-
*pSB4K5(EcoR1, Pst1) 9.6μg/mL 1.28(260/280) 0.32(260/230)
+
-
====Miniprep====
+
-
*J23107-tatABCD-psp-DT(pSB1C3) 78μg/mL
+
-
*J23107-tatABCD-psp-DT(pSB1C3) 48μg/mL
+
-
*LacP-torA-GFP-DT 29μg/mL
+
-
*J23101-kil-DT 25μg/mL
+
-
 
+
-
====Restrictive Digestion====
+
-
 
+
-
{| class="wikitable"
+
-
!J23101-Kil-DT 25µg/ml||BufferM||Xba1||Pst1||BSA||MilliQ||Total
+
-
|-
+
-
|40||6||1||1||6||6||60
+
-
|}
+
-
{| class="wikitable"
+
-
!J23101-Kil-DT 25µg/ml||BufferM||Xba1||BSA||MilliQ||Total
+
-
|-
+
-
|3||1||0.5||1||4.5||10
+
-
|}
+
-
{| class="wikitable"
+
-
!J23101-Kil-DT 25µg/ml||BufferH||Pst1||MilliQ||Total
+
-
|-
+
-
|3||1||0.5||5.5||60
+
-
|}
+
-
at 37℃ for 2 hours
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis091801.jpg|200px|right]]
+
-
①. pSB4K5<br>
+
-
②. pSB4K5(Pst1)<br>
+
-
③. pSB4K5(EcoR1)<br>
+
-
④. pSB4K5(EcoR1, Pst1)<br>
+
-
⑤. LacP-torA-GFP-DT<br>
+
-
⑥. LacP-torA-GFP-DT(Xba1)<br>
+
-
⑦. LacP-torA-GFP-DT(Pst1)<br>
+
-
⑧. LacP-torA-GFP-DT(Xba1, Pst1)<br><br>
+
-
 
+
-
[[File:Electrophoresis091802.jpg|200px|right]]
+
-
①. 1kb ladder<br>
+
-
②. J23101-Kil-DT(Xba1, Pst1)<br>
+
-
③. J23101-Kil-DT(Xba1, Pst1)<br>
+
-
④. J23101-Kil-DT(Xba1, Pst1)<br>
+
-
⑤. J23101-Kil-DT(Xba1, Pst1)<br>
+
-
⑥. 1kb ladder<br>
+
-
 
+
-
====Restrictive Digestion====
+
-
 
+
-
{| class="wikitable"
+
-
!J23101-Kil-DT||BufferM||Xba1||Pst1||BSA||MilliQ||Total
+
-
|-
+
-
|15||3||1||1||3||7||30
+
-
|}
+
-
 
+
-
{| class="wikitable"
+
-
!J23101-Kil-DT||BufferM||Xba1||BSA||MilliQ||Total
+
-
|-
+
-
|3||1||0.5||1||4.5||10
+
-
|}
+
-
 
+
-
{| class="wikitable"
+
-
!J23101-Kil-DT||BufferH||Pst1||MilliQ||Total
+
-
|-
+
-
|3||1||0.5||5.5||10
+
-
|}<br>
+
-
37℃<br>
+
-
 
+
-
==September 19==
+
-
====Gel extraction====
+
-
lane1. 1kb ladder<br>
+
-
lane2. pSB4K5(EcoR1, Pst1)<br>
+
-
lane3. pSB4K5(EcoR1, Pst1)<br>
+
-
lane4. empty<br>
+
-
lane5. LacP-torA-GFP-DT(Xba1, Pst1)<br>
+
-
lane6. LacP-torA-GFP-DT(Xba1, Pst1)<br>
+
-
[[File:GelEx0919.jpg|200px]]
+
-
[[File:GelEx091901.jpg|200px]]
+
-
 
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!LacP-torA-GFP-DT(Xba1,Pst1) 9.7μg/mL||pSB4K5(EcoR1,Pst1) 9.6μg/mL||J23107-tatABCD-PsPA-DT(EcoR1,Spe1) 14.2μg/mL||Ligation High Ver.2||Total
+
-
|-
+
-
|7||2||7||8||24μL
+
-
|}
+
-
{| class="wikitable"
+
-
!pSB4K5(EcoR1,Pst1) 9.6μg/mL||Ligation High Ver.2||MilliQ||Total
+
-
|-
+
-
|2||1||21||24μL
+
-
|}<br>
+
-
incubate at 37℃ for 2h
+
-
====Electrophoresis====
+
-
[[File:CheckingElectrophoresis0919.jpg|200px|thumb|right]]
+
-
J23101-kil-DT (Xba1,Pst1), (Xba1), (Pst1)<br>
+
-
Lane1: 1kb ladder<br>
+
-
Lane2: empty<br>
+
-
Lane3: plasmid<br>
+
-
Lane4: (Xba1)<br>
+
-
Lane5: (Pst1)<br>
+
-
Lane6: (Xba1, Pst1)<br>
+
-
 
+
-
====Liquid Culture====
+
-
We did liquid culture J23106-kil-DT(9/10, Amp)'s 6,7 at LB mediumAmp.
+
-
====Transformation====
+
-
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT<br>
+
-
pSB4K5 negative control<br>
+
-
====Liquid culture====
+