Team:KAIT Japan/Notebook

From 2011.igem.org

(Difference between revisions)
 
(One intermediate revision not shown)
Line 12: Line 12:
font-size: x-large;
font-size: x-large;
font-family: "Gill Sans Ultra Bold";
font-family: "Gill Sans Ultra Bold";
-
color: #0000FF;
+
color: #008000;
}
}
.style3 {
.style3 {
Line 18: Line 18:
font-family: "Gill Sans", "Gill Sans MT", Calibri, "Trebuchet MS", sans-serif;
font-family: "Gill Sans", "Gill Sans MT", Calibri, "Trebuchet MS", sans-serif;
margin-left: 40px;
margin-left: 40px;
 +
color: #FF0000;
}
}
.style4 {
.style4 {
Line 52: Line 53:
border-top-width: 1px;
border-top-width: 1px;
padding: 1px 4px;
padding: 1px 4px;
 +
}
 +
.style14 {
 +
color: #FF0000;
}
}
</style>
</style>
Line 57: Line 61:
<body>
<body>
-
 
+
<h2><span id="Protocol" class="style1">Protocol</span></h2>
-
<p class="style1"><strong class="style2">Protocol</strong></p>
+
<p class="style3"><strong>PCR</strong></p>
-
<p class="style3">PCR</p>
+
<p class="style4">1.Add 100μL of reagent solution(Table) to microtube. <br />
<p class="style4">1.Add 100μL of reagent solution(Table) to microtube. <br />
2.Dispense 20μL PCR solution to PCR tube. <br />
2.Dispense 20μL PCR solution to PCR tube. <br />
Line 121: Line 124:
</p>
</p>
<p>&nbsp;</p>
<p>&nbsp;</p>
-
<p class="style3">Transformation <br />
+
<p class="style3"><strong>Transformation </strong> <br />
-
 Preparation of E. coli contain particular plasmid </p>
+
<strong> Preparation of E. coli contain particular plasmid </strong> </p>
<p class="style4">1.Incubate frozen competent cell on the ice. <br />
<p class="style4">1.Incubate frozen competent cell on the ice. <br />
2.Add 2μL of plasmid to competent cell. <br />
2.Add 2μL of plasmid to competent cell. <br />
Line 132: Line 135:
8.Incubate over night at 37°C. </p>
8.Incubate over night at 37°C. </p>
<p>&nbsp;</p>
<p>&nbsp;</p>
-
<p class="style3">Restriction enzyme digestion of DNA </p>
+
<p class="style3"><strong>Restriction enzyme digestion of DNA </strong> </p>
<p class="style4">1.Mix DNA and restriction enzyme(Table). <br />
<p class="style4">1.Mix DNA and restriction enzyme(Table). <br />
2.Incubate over night at 37°C. <br />
2.Incubate over night at 37°C. <br />
Line 168: Line 171:
</p>
</p>
<p>&nbsp;</p>
<p>&nbsp;</p>
-
<p class="style3">Plasmid extraction<br />
+
<p class="style3"><span class="style14"><strong>Plasmid extraction</strong></span><br class="style14" />
-
 Preparation of plasmid extracted from E. coli </p>
+
<span class="style14"><strong> Preparation of plasmid extracted from E. coli  
 +
</strong></span> </p>
<p class="style4">1. Pick up single colony from agar plate and cultivate it in  
<p class="style4">1. Pick up single colony from agar plate and cultivate it in  
20 mL LB medium containing&nbsp;&nbsp;&nbsp; <br />
20 mL LB medium containing&nbsp;&nbsp;&nbsp; <br />

Latest revision as of 17:07, 5 October 2011

無題 2

Home Team Official Project Notebook Safety Sponsors

無題 1

Protocol

PCR

1.Add 100μL of reagent solution(Table) to microtube.
2.Dispense 20μL PCR solution to PCR tube.
3.Amplifty target DNA with PCR program.
4.Confirm the band of DNA by agar gel electrophoresis.

reagent name

Volume(μL)
TaKaRa Ex Taq 0.5

10×Ex Taw buffer

10

dNDP Mixture

8

template DNA

2

fowerd primer

4

reverse primer

4
sterile water 71.5
total 100

 

Transformation
 Preparation of E. coli contain particular plasmid

1.Incubate frozen competent cell on the ice.
2.Add 2μL of plasmid to competent cell.
3.Incubate for 15 minutes on the ice.
4.Incubate for 45 seconds at 42°C.
5.Incubate for 2 minutes on the ice.
6.Add 250μL LB medium and cultivate for 1 hour at 37°C.
7.Spread culture medium on LB agar plate with appropriate antibiotic.
8.Incubate over night at 37°C.

 

Restriction enzyme digestion of DNA

1.Mix DNA and restriction enzyme(Table).
2.Incubate over night at 37°C.
3.Confirm the band of DNA by agar gel electrophoresis.

reagent name Volume(μL)
DNA 15
EcoR I 1
Xba I 1
buffer M 2
steril water 1
total 20

 

Plasmid extraction
 Preparation of plasmid extracted from E. coli

1. Pick up single colony from agar plate and cultivate it in 20 mL LB medium containing   
  appropriate antibiotic overnight at 37°C.
2. Move the culture medium to 50 mL falcon.
3. Centrifuge for 5 minutes at 6000rpm and 4 °C and discard solution.
4. Add 2 mL Solution1, the pellet.
5. Add 4 mL Solution 2, invert tube and stored 3 minutes on ice.
6. Add 3 mL Solution 3, invert tube and stored 5 minutes on ice.
7. Centrifuge for 10 minutes at 9,500rpm and 4 °C.
10.Take aqueous layer to new 50 mL falcon.
11.Add 40 μL RNase, invert tube and incubate for 30 minutes.
12.Add 2 mL phenol chloroform mixture.
13.Centrifuge for 5 minutes at 9,500rpm and 4 °C. 
14.Take supernatant to new 50 mL falcon.
15.Add 3 mL chloroform solution and misce.
16.Centrifuge for 3 minutes at 9,500rpm and 4 °C.
17.Take 3 mL supernatant to new 50 mL falcon.
18.Add 300 μL sodium acetate.
19.Add 7.5 mL 100%ethanol.
20.Centrifuge for 20 minutes at 9,500rpm and 4 °C and discard solution.
21.Add 8 mL 70% ethanol.
22.Centrifuge for 20 minutes at 9,500rpm and 4 °C and discard solution.
23.Add 50 μL TE buffer and tapping.
24.Preserve at freezer.