Team:HokkaidoU Japan/Notebook

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(Liquid culturing 16hrs->15hrs 30min, mini-prep, Electrophoresis)
 
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{{Team:HokkaidoU_Japan/header}}
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!align="center"|[[Team:HokkaidoU_Japan|Home]]
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{{Team:HokkaidoU_Japan/nav.notebook}}
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!align="center"|[[Team:HokkaidoU_Japan/Team|Team]]
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<div id="hokkaidou-column-main">
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!align="center"|[https://igem.org/Team.cgi?year=2012&team_name=HokkaidoU_Japan Official Team Profile]
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<!-- DO NOT EDIT OVER THIS LINE @iTakeshi -->
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!align="center"|[[Team:HokkaidoU_Japan/Project|Project]]
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!align="center"|[[Team:HokkaidoU_Japan/Modeling|Modeling]]
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    .hokkaidou-notebook-index {
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!align="center"|[[Team:HokkaidoU_Japan/Notebook|Notebook]]
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!align="center"|[[Team:HokkaidoU_Japan/Safety|Safety]]
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!align="center"|[[Team:HokkaidoU_Japan/Attributions|Attributions]]
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== Hello ==
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We are team HokkaidoU Japan! Today we learn and start to edit wiki.
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(>ω<)　<br>
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Dear Mr.Ortiz, I saw the help page which you edited.<br>
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    }
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hola!<br>
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    .hokkaidou-notebook-index h5 {
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        font-size:24px;
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==March==
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        line-height:1;
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===Spring Boot Camp===
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        padding:0 0 10px 0;
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;date
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    }
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:March 5 (Mon) ~ March 9 (Fri)
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    .hokkaidou-notebook-index h5 {
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====Monday, March 5====
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        color:white;
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;Session #1
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    }
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:Short lecture about moleculer biology (Mr.Yamazaki, our adviser)
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    .hokkaidou-notebook-index:hover p {
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;Session #2
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        color:white;
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:Tutrial: How to use 'Unipro UGENE' (iTakeshi)
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    }
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;Session #3
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    .hokkaidou-notebook-index p {
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:Guidance: Wiki Reading (Laury)
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        font-size:12px;
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::Example: 2010 MIT
+
        width:500px;
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====Tuesday, March 6====
+
        line-height:1.5;
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;Session #4~6
+
        color: #ffffff;
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:Reading Wikis in turn and discussions
+
    }
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:*2010 NYU
+
    .hokkaidou-notebook-index:hover {
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:*2009 Cambridge
+
        background-position:200px 50%;
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:*2009 Growningen
+
    }
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====Wednesday, March 7====
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    #hokkaidou-notebook-bootcamp {
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;Session #7~11
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        background-image: url('https://static.igem.org/mediawiki/2012/e/ea/HokkaidoU2012_Menu_Circle.png');
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:Reading Wikis (2)
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        background-position: 673px -522px;
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:*2010 Washington
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    }
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:*2009 Valencia
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    #hokkaidou-notebook-bootcamp:hover {
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:*2011 Barklay
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:*2010 Paris
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:*2010 Bristol
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    }
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====Thursday, March 8====
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    #hokkaidou-notebook-diary {
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;Session #12
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:2012 Project Brainstorming
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        background-position: 673px -653px;
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::The details is secret! :)
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    }
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;Session #13
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    #hokkaidou-notebook-diary:hover {
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:Guidance: How to read papers (Laury)
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        background-image: url('https://static.igem.org/mediawiki/2012/e/ea/HokkaidoU2012_Menu_Circle.png');
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====Friday, March 9====
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        background-position: 543px -653px;
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;Session #14
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    }
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:2012 Project Brainstorming (2)
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    #hokkaidou-notebook-protocols {
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;Session #15
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        background-image: url('https://static.igem.org/mediawiki/2012/e/ea/HokkaidoU2012_Menu_Circle.png');
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:Guidance: How to look up papers you want (Laury)
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        background-position: 673px -783px;
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;Session #16
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    }
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:Tutorial: Modeling the behavior of cells (iTakeshi)
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    #hokkaidou-notebook-protocols:hover {
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;Session #17
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        background-image: url('https://static.igem.org/mediawiki/2012/e/ea/HokkaidoU2012_Menu_Circle.png');
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:Final Session: Reviewing this camp
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        background-position: 543px -783px;
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;Party!!
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    }
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</style>
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==Experiment Calender==
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<h2>Notebook</h2>
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{|class="calendar"
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<div class="hokkaidou-section">
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|-
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<a href="/Team:HokkaidoU_Japan/Notebook/Spring_Boot_Camp" class="hokkaidou-notebook-index" id="hokkaidou-notebook-bootcamp">
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|colspan="7"|July
+
  <h5>Boot Camp</h5>
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|-
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  <p>Be my apprentice.</p>
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!style="color:red;"|S
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</a>
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!M
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<a href="/Team:HokkaidoU_Japan/Notebook/Lab_Diary" class="hokkaidou-notebook-index" id="hokkaidou-notebook-diary">
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!T
+
  <h5>Lab Diary</h5>
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!W
+
  <p>That's one small step for you, one giant leap for us. </p>
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!T
+
</a>
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!F
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<a href="/Team:HokkaidoU_Japan/Notebook/overall_protocols" class="hokkaidou-notebook-index" id="hokkaidou-notebook-protocols">
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!style="color:blue;"|S
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  <h5>Protocols</h5>
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|-
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  <p>Bible of our lab.</p>
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|style="color:red;"|1
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</a>
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|2
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</div>
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|3
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</html>
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|4
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<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi -->
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|5
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</div>
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|6
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=July=
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==phaABC team==
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-
 
+
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now experimenting...
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-
 
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==Ag43&Lysis team==
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===week 1(4th~10th)===
+
-
 
+
-
----
+
-
*4th
+
-
 
+
-
;Transformation
+
-
Transformation of BBa_B0015(dT), B0034(RBS), I179005(pT7), K346007(Ag43), and K542009(pLacI-RBS-Ag43) in DH5α
+
-
#Added each DNA solutions (1ul) into DH5α comeptent cell and standed on ice in 30 min.
+
-
#Cultivated on LBA(dt,RBS,T7) and LBC(Ag43, pLacI-RBS-Ag43). E.coli cultivate in  LBC was pre-cultivated in 2 hrs. pT7 was 21hrs and Others were 20hrs cultivated
+
-
<br>
+
-
 
+
-
----
+
-
*5th
+
-
 
+
-
;Transformation
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-
K346007(Ag43) was failed to cultivate on LBC plate.
+
-
Transformation of K346007(Ag43) in DH5α.
+
-
#Add DNA solution(1ul) into DH5α comeptent cell and stand on ice in 30 min.
+
-
#Pre-cultivated in 2hrs.
+
-
#Cultivated on LBC in 21hrs.
+
-
 
+
-
;Single colony isolation
+
-
Single colony isolation of BBa_B0015, B0034, I179005 and K542009.
+
-
#Picked up one colony.
+
-
#Cultivation on LBA(dt,RBS,T7) and LBC(pLacI-RBS-Ag43) in 14hrs30mins
+
-
BBa_K542009 was Ag43 only part! And the part didn't have Biobrick suffix.
+
-
 
+
-
 
+
-
----
+
-
*6th
+
-
 
+
-
;Liquid culture
+
-
Liquid culture in LBA(dT,RBS,pT7) and LBC(pLacI-RBS-Ag43)
+
-
#Picked up two colonies from each plates.
+
-
#One colony was dipped in 1ml LB (A or C), and other colony was dipped in 2ml LB(A or C). 1ml is for glycerol stocks and 2ml is for mini-prep.
+
-
#16hrs Cultivation
+
-
<br>
+
-
;Single colony isolation
+
-
#Single colony isolation of K346007(Ag43).
+
-
<br>
+
-
 
+
-
----
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-
*7th
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-
 
+
-
 
+
-
;Liquid culture
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-
Liquid culture in LBC(Ag43).
+
-
#Picked up two colonies from each plates.
+
-
#Both of colonies were dipped in 2ml LBC, and then we cultivate them in 38℃.<br>
+
-
However, one of them cultivated only 8 hours. It's for glycerol stock.
+
-
<br>
+
-
 
+
-
 
+
-
'''3A assembly!'''<br>
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-
Assembled pT7, RBS and pSB1C3 by 3A assembly.
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-
This 3A assembly is our first try!
+
-
 
+
-
;mini-prep
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#mini-prep of dT,RBS,pT7 and pLacI-RBS-Ag43.
+
-
#Elution in 50ul buffer
+
-
<br>
+
-
 
+
-
;Glycerol stock
+
-
Made glycerol stocks of dT,RBS,pT7 and pLacI-RBS-Ag43.
+
-
#Parts written above were cultivated in LBA(dT,RBS,pT7) and LBC(pLacI-RBS-Ag43) 1ml in 16hrs 30min.
+
-
#Add glycerol and Freeze at -80C
+
-
<br>
+
-
 
+
-
[[Image:120707_I719005_B0034_B0015_K542009_mini-prep_umeuchi.jpg]]
+
-
;Electrophoresis
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-
Electrophoresis to predict concentration of mini-prep products(dT,RBS,pT7 and pLacI-RBS-Ag43).
+
-
#Used 1% agarose gel.
+
-
#Pre-migration.
+
-
#Migrated 1.2ul of DNA solutions (1ul is mini-prep products and 0.2ul is Loading Dye) in 35min.
+
-
#Took a photograph of 1% agarose gel that finished electrophoresis.
+
-
<br>
+
-
 
+
-
;Digestion
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-
<br>
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-
Digestion of I719005, B0034 and pSB1K3
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-
 
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Digestion recipe
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All parts were reacted in 30ul solution.
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*I719005(40ng/ul)
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{|
+
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|DNA solution
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|12.5ul
+
-
|-
+
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|EcoRI
+
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|1ul
+
-
|-
+
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|SpeI
+
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|1ul
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-
|-
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|10xH Buffer
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|3ul
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|-
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|DW
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|12.5ul
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-
|}
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+
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*B0034(40ng/ul)
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{|
+
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|DNA solution
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|12.5ul
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|-
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|XbaI
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|1ul
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|-
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|PstI
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|1ul
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|-
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|10xM Buffer
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|3ul
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|-
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|DW
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|12.5ul
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-
|}
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+
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*pSB1K3(25ng/ul)
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{|
+
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|DNA solution
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|12ul
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-
|-
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|EcoRI
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|1ul
+
-
|-
+
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|PstI
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|1ul
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-
|-
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|10xH Buffer
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|3ul
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-
|-
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|DW
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|13ul
+
-
|}
+
-
<br>
+
-
;Ethanol precipitation
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For rising concentration of DNA solution which use for Ligation and removing restriction enzyme.
+
-
#Added 3ul of NaoAc, 1.5ul of glycogen and 75ul of 100% ethanol.
+
-
#Centrifuged in 14000rpm, 30min at 4C.
+
-
#Remove supernatant and added 220ul of 70% ethanol.
+
-
#Centrifuged in 15000rpm, 15min at 4C.
+
-
#Remove supernatant and air drying in room temperature then added 10ul of DW.
+
-
<br>
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;Ligation
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All DNA solutions were digested.
+
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3A assembly protocol required Ligation reaction should be in total 25ul solution.
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{|
+
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|Ligation Mighty Mix
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|12.5ul
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|-
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|pT7
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|2ul
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|-
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|RBS
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|2ul
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|-
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|pSB1K3
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|2ul
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|-
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|DW
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|6.5ul
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|-
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|――――――――――
+
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|-
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|Total
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|25ul
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|}
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-
 
+
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Ligation reaction recipe was written below.
+
-
 
+
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{|
+
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|Degree
+
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|Minute
+
-
|-
+
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|16
+
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|30
+
-
|-
+
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|65
+
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|10
+
-
|-
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|4
+
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|Hold
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-
|}
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<br>
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ligation was finished.
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But now pm10:00. 2.5hrs needs for doing transformation. Transformation would finish at am:0:30.
+
-
 
+
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Withdraw!!!!
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-
 
+
-
----
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*8th
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-
 
+
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*(pT7 + RBS)
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;Transformation
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-
Transformation for pT7+RBS+pSB1K3
+
-
#Added DNA soltions (Ligation products) 1ul to DH5α compitent cell.
+
-
#Stood on ice in 30min.
+
-
#Added 600ul of LB to transformed DH5α solution.
+
-
#Pre-cultivate in 2hrs
+
-
#Splead 300ul of LB&DH5α solution to LBK.
+
-
#Cultivated 
+
-
<br>
+
-
 
+
-
*K346007(Ag43)
+
-
;mini-prep
+
-
mini-prep for Liquid culture product of K346007(Ag43)
+
-
#Used FastGene Plasmid Mini Kit(Nippon Genetics)
+
-
#Elutioned in 50ul
+
-
#First we eluted in colection tube. then moved in Eppendorf tube.
+
-
 
+
-
 
+
-
;Erectrophoresis
+
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Erectrophoresis for mini-prep product(Ag43).
+
-
#Prepared 1% Agalose gel and added EtBr then pre-migration in 30min.
+
-
#1ul 1kb ladder, 1.2ul mini-prep product(1ul is DNA solution and 0.2ul is loading dye) added then migtrated
+
-
 
+
-
mini-prep result (With ligation result of pT7+RBS+pSB1K3)
+
-
 
+
-
[[image:HokkaidoU2012 120708 K346007 miniprep and pt7 rbs.jpg]]
+
-
 
+
-
;Glycerol stock
+
-
Made glycerol stock of K346007 (Ag43).
+
-
#Parts written above were cultivated in LBC.
+
-
#Added glycerol and Freezed at -80C
+
-
 
+
-
 
+
-
*(Ag43 + dT)
+
-
Assembling K346007(Ag43) + B0015(dT) with 2-piece assembly(Biobrisk standard assembly)
+
-
 
+
-
 
+
-
;Digestion
+
-
Digested Ag43 and dT in solution by recipes Written below.
+
-
Insert DNA required too much weight and volume(volume was calculated from concentration of DNA mini-prep
+
-
product)from our calculation. There are no insurance of succession of digestion.
+
-
+
-
*Ag43(Insert)
+
-
5190bp(Ag43 + pSB1C3)
+
-
{|
+
-
|DNA solution
+
-
|48ul
+
-
|-
+
-
|EcoRI
+
-
|1ul
+
-
|-
+
-
|SpeI
+
-
|1ul
+
-
|-
+
-
|10xH buffer
+
-
|6ul
+
-
|-
+
-
DW
+
-
|4ul
+
-
|-
+
-
|――――――――――――
+
-
|-
+
-
|Total
+
-
|60ul
+
-
|}
+
-
 
+
-
 
+
-
*dT(Vector)
+
-
3318bp(Ag43 + pSB1AK3)
+
-
{|
+
-
|DNA solution
+
-
|8ul
+
-
|-
+
-
|EcoRI
+
-
|1ul
+
-
|-
+
-
|XbaI
+
-
|1ul
+
-
|-
+
-
|10xM buffer
+
-
|2ul
+
-
|-
+
-
|DW
+
-
|8ul
+
-
|-
+
-
|――――――――――――
+
-
|-
+
-
|Total
+
-
|20ul
+
-
|}
+
-
 
+
-
 
+
-
Digestion result image
+
-
 
+
-
 
+
-
[[image:HokkaidoU2012 120708 dt K346007 digestion umeuchi.jpg]]
+
-
 
+
-
 
+
-
K346007(Ag43) was 5190bp before digestion (Biobrick prefix + Ag43 + Biobrick suffix + pSB1C3).
+
-
After digestion, Ag43 and pSB1C3 was separated and became fragments about 3120bp(Ag43) and 2070bp(pSB1C3).
+
-
Gel image above shows there are two fragments, one fragment is about 2000bp other fragment is 3000bp.
+
-
Digestion would be succeeded.
+
-
About Vector(Boo15:dT), the DNA was circular so DNA migrated more far than Linear DNA. d+ (Circular DNA) migrated little more far than d- (Linear DNA). so we think digestion was succeeded.
+
-
 
+
-
 
+
-
;Ethanol precipitation
+
-
Ethanol precipitation for gel extraction products(K346007(Ag43) and B0015(dT))
+
-
#Added 5ul of NaoAc , 1.5ul of glycogen and 125ul of 100% ethanol to 50ul DNA solutions.
+
-
#Centrifuged in 15000rpm, 10min at 4C.
+
-
#Remove supernatant and added 220ul of 70% ethanol.
+
-
#Centrifuged in 15000rpm, 5min at 4C.
+
-
#Remove supernatant and air drying in room temperature then added 10ul of DW.
+
-
 
+
-
;Ligation
+
-
All DNA solutions were digested.
+
-
Used Ligation Mighty Mix(TakaraBio)
+
-
 
+
-
{|
+
-
|Ligation Mighty Mix
+
-
|5ul
+
-
|-
+
-
|Insert: Ag43
+
-
|2ul
+
-
|-
+
-
|Vector: dT
+
-
|2ul
+
-
|-
+
-
|DW
+
-
|1ul
+
-
|-
+
-
|――――――――――
+
-
|-
+
-
|Total
+
-
|10ul
+
-
|}
+
-
 
+
-
 
+
-
Ligation reaction recipe was written below.
+
-
 
+
-
{|
+
-
|Degree
+
-
|Minute
+
-
|-
+
-
|16
+
-
|30
+
-
|-
+
-
|65
+
-
|10
+
-
|-
+
-
|4
+
-
|Hold
+
-
|}
+
-
 
+
-
 
+
-
;Electrophoresis
+
-
Confirmation of succession of ligation.
+
-
#Prepared 1% Agalose gel and added EtBr then pre-migration in 30min.
+
-
#Added 1kb ladder, Ligation product(1ul) and digestion products (control:each solutions 1ul).
+
-
#Migtrated in 30min.
+
-
 
+
-
Electrophoresis results
+
-
 
+
-
 
+
-
[[image:HokkaidoU2012 120708 K346007-dT ligation.jpg]]
+
-
 
+
-
 
+
-
;Transformation
+
-
Transformation for K346007(Ag43)+B0015(dT) on pSB1AK3.
+
-
#Added DNA soltions (Ligation products) 1ul to DH5α compitent cell.
+
-
#Stood on ice in 30min.
+
-
#Added 600ul of LB to transformed DH5α solution.
+
-
#From 700 solution(100ul is DH5α and 600ul is LB), 100ul add to 900ul of LB(x10 solution)
+
-
#Spread 300ul from 600(700-100)ul and 1000ul of LB&DH5α solution to each LBA plates.
+
-
#Cultivated.
+
-
 
+
-
----
+
-
*9th
+
-
 
+
-
pT7 + RBS (3A Assembly) and  Ag43 + dT (standard assembly) ligation products were transformed and cultivated then some colonies were existed (12~16 colonies) so we confirmed really insert DNA (3A:pT7 and RBS, standard:Ag43 and dT) were inserted to vector or not by colony PCR.
+
-
;Colony PCR
+
-
Colony PCR for assembly products.Each product reacted recipes written below.
+
-
#picked up each 12(16 is best but there were only 12 colonies) colonies from LB plates by Autoclaved toothpicks.
+
-
#Dipped into 10ul DW in 1.5ml eppendorf tubes.
+
-
#from 10ul DW, 4ul was added in PCR reaction solution (below), 6ul was mixed with 200ul LB.
+
-
#Ran PCR machine in recipe below.
+
-
#Electrophoresis for confirmation of PCR results.
+
-
 
+
-
 
+
-
PCR reaction solution
+
-
{|
+
-
|DNA solution
+
-
|4ul
+
-
|-
+
-
|KapaTaq ready mix
+
-
|5ul
+
-
|-
+
-
|BioBrick prefix forward primer
+
-
|0.5ul
+
-
|-
+
-
|BioBrick suffix reverse primer
+
-
|0.5ul
+
-
|-
+
-
|――――――――――――――――――――――――――
+
-
|-
+
-
|Total
+
-
|10ul
+
-
|}
+
-
 
+
-
 
+
-
'''PCR recipe'''
+
-
 
+
-
(pT7 + RBS)
+
-
{|
+
-
|Number
+
-
|Degree
+
-
|Second
+
-
|-
+
-
|1
+
-
|94
+
-
|120
+
-
|-
+
-
|2
+
-
|94
+
-
|30
+
-
|-
+
-
|3
+
-
|68
+
-
|60
+
-
|-
+
-
|4
+
-
|4
+
-
|HOLD
+
-
|}
+
-
Cycle:2~3 x 40
+
-
 
+
-
 
+
-
(Ag43 + dT)
+
-
 
+
-
Ag43 + dT (+ Biobrick prefis & suffix) is about 3290bp. Extension step needed >180seconds.
+
-
{|
+
-
|Number
+
-
|Degree
+
-
|Second
+
-
|-
+
-
|1
+
-
|94
+
-
|120
+
-
|-
+
-
|2
+
-
|94
+
-
|30
+
-
|-
+
-
|3
+
-
|68
+
-
|180
+
-
|-
+
-
|4
+
-
|4
+
-
|HOLD
+
-
|}
+
-
Cycle:2~3 x 35
+
-
 
+
-
 
+
-
;Electrophoresis results
+
-
Electrophoresis for (pT7 + RBS) in 2% agarose gel and (Ag43 + dT) in 1% agarose gel.
+
-
 
+
-
 
+
-
pT7 + RBS on pSB1K3
+
-
bbp-Insert-bbs:86bp
+
-
 
+
-
[[image:HokkaidoU2012 120709 pt7-rbs 75% scale.jpg]]
+
-
 
+
-
 
+
-
Ag43 + dT on pSB1AK3
+
-
bbp-Insert-bbs:3290bp
+
-
 
+
-
[[image:HokkaidoU2012 120709 ag43-dt-1 75% scale.jpg]]
+
-
 
+
-
 
+
-
We couldn't confirm insert DNA were really ligated with Vector or not.
+
-
Next we tried confirmation of insert DNA by Electrophoresis of mini-prep products.  
+
-
For mini-prep, we needed do liquid culture.
+
-
 
+
-
 
+
-
;Liquid culturing
+
-
Liquid culture for mini-prep((pT7 + RBS) on pSB1K3 and (Ag43 + dT) on pSB1AK3).
+
-
#Prepared 1800ul LBK(for (pT7 + RBS) on pSB1K3) and LBA(for (Ag43 + dT) on pSB1AK3).
+
-
#Added 200ul of LB solutions (colony PCR solutions were pre-cultivated in about 2hrs).
+
-
#Cultivated 15hrs30min.
+
-
 
+
-
----
+
-
*10th
+
-
 
+
-
 
+
-
;Mini-prep
+
-
Mini-prep for some colonies (we selected colonies which showed more correct bands than other colonies, pT7+RBS were No.4, 5, 9, 10 colonies and Ag43 + dT were No.1, 2, 3, 4 colonies)cultivated in LBA(Ag43 + dT) and LBK(pT7 + RBS) in 15hrs 30min.
+
-
#Mini-prep for LB culturing products along the protocol of FastGene Plasmid Mini kit(Nippon Genetics).
+
-
#Electrophoresis (Used pre-migrated 1% agarose gels with 5ul EtBr)in 30min.
+
-
 
+
-
Electrophoresis resulsts
+
-
 
+
-
pT7 + RBS on pSB1K3(Total 2247bp)
+
-
[[image:]]
+
-
 
+
-
 
+
-
Ag43 + dT on pSB1AK3(Total 6444bp)
+
-
[[image:]]
+

Latest revision as of 00:52, 27 September 2012

Notebook

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Lab Diary

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Protocols

Bible of our lab.


Retrieved from "http://2012.igem.org/Team:HokkaidoU_Japan/Notebook"