Team:TU Darmstadt/protocols/InFusion

From 2013.igem.org

(Difference between revisions)
 
(6 intermediate revisions not shown)
Line 2: Line 2:
<style type="text/css">
<style type="text/css">
-
body
+
body  
{
{
margin:0;  
margin:0;  
Line 15: Line 15:
{
{
   background-color: white;
   background-color: white;
-
   background-image: url("/wiki/images/7/70/Hintergrund3.jpg");
+
   background-image: url("/wiki/images/5/5c/Hintergrund_Grün(ohne_Header%2C_Logo_und_Schlagschatten).jpg");
   background-attachment: scroll;
   background-attachment: scroll;
   background-position: 50% 0;
   background-position: 50% 0;
Line 26: Line 26:
{
{
     background: white;
     background: white;
-
     background-image: url('/wiki/images/2/2f/Header_(mit_Logo).jpg');
+
     background-image: url("/wiki/images/2/2f/Header_%28mit_Logo%29.jpg");
     margin: 0 auto;
     margin: 0 auto;
     height:250px ;
     height:250px ;
Line 35: Line 35:
     background-repeat: no-repeat;
     background-repeat: no-repeat;
     background-size: 100% 100% ;
     background-size: 100% 100% ;
-
     border-style: none;
+
     border-style: none;;}
-
}
+
#p-logo { display:none;}
#p-logo { display:none;}
Line 42: Line 41:
p {line-height:1.5em; margin:0 0 15px; text-align:justify; color:white;}
p {line-height:1.5em; margin:0 0 15px; text-align:justify; color:white;}
h1 {font-size:1.8em; font-weight:400; margin:0 0 12px;}
h1 {font-size:1.8em; font-weight:400; margin:0 0 12px;}
 +
#mm_icon1
#mm_icon1
{
{
position: absolute;
position: absolute;
-
top: 240px;  
+
top: 150px;  
-
left: 200px;
+
left: 30px;
}
}
#mm_icon2
#mm_icon2
{
{
-
position: relative;
+
position: absolute;
-
top: 170px;  
+
top: 150px;  
left: 350px;
left: 350px;
}
}
Line 67: Line 67:
{
{
position: absolute;
position: absolute;
-
top: 260px;  
+
top: 150px;  
-
left: 230px;
+
left: 30px;
background:white;
background:white;
filter:alpha(opacity=83); opacity:0.83;
filter:alpha(opacity=83); opacity:0.83;
Line 80: Line 80:
{
{
position: absolute;
position: absolute;
-
top: 670px;  
+
top: 150px;  
left: 350px;
left: 350px;
background:white;
background:white;
Line 89: Line 89:
border-radius:15px;
border-radius:15px;
}
}
 +
 +
#abstracticon3
#abstracticon3
Line 94: Line 96:
position: absolute;
position: absolute;
top: 150px;  
top: 150px;  
-
left: 700px;
+
left: 670px;
background:white;
background:white;
filter:alpha(opacity=83); opacity:0.83;
filter:alpha(opacity=83); opacity:0.83;
Line 102: Line 104:
border-radius:15px;
border-radius:15px;
}
}
-
 
#taskbar
#taskbar
Line 108: Line 109:
position:absolute;
position:absolute;
top:10px;
top:10px;
-
left:400px;
+
left:500px;
z-index: 5;
z-index: 5;
}
}
-
</style>
 
 +
 +
 +
dl.igemTUD2013gelpicture
 +
{
 +
border: 1px solid #000;
 +
background-color: #109f71;
 +
width: 110px;
 +
text-align: center;
 +
padding: 5px 5px 5px 5px;
 +
float: right;
 +
margin: 0 0 0 0;
 +
margin-left:15px
 +
}
 +
 +
.igemTUD2013gelpicture dt
 +
{
 +
font-weight: bold;
 +
background-color: #131210;
 +
color: #959289;
 +
padding: 0 0;
 +
margin-bottom: 10px;
 +
}
 +
 +
.igemTUD2013gelpicture dd img
 +
{
 +
border: 1px solid #000;
 +
width: 100px;
 +
height: 200px;
 +
}
 +
 +
.igemTUD2013gelpicture dd
 +
{
 +
margin: 0;
 +
padding: 5px 5px 5px 5px;
 +
font-size: 100%;
 +
text-align: left;
 +
}
 +
 +
 +
 +
 +
dl.igemTUD2013gelpicture2
 +
{
 +
border: 1px solid #000;
 +
background-color: #109f71;
 +
width: 210px;
 +
text-align: center;
 +
padding: 5px 5px 5px 5px;
 +
float: right;
 +
margin: 0 0 0 0;
 +
margin-left:15px
 +
}
 +
 +
.igemTUD2013gelpicture2 dt
 +
{
 +
font-weight: bold;
 +
background-color: #131210;
 +
color: #959289;
 +
padding: 0 0;
 +
margin-bottom: 10px;
 +
}
 +
 +
.igemTUD2013gelpicture2 dd img
 +
{
 +
border: 1px solid #000;
 +
width: 200px;
 +
height: 200px;
 +
}
 +
 +
.igemTUD2013gelpicture2 dd
 +
{
 +
margin: 0;
 +
padding: 5px 5px 5px 5px;
 +
font-size: 100%;
 +
}
 +
LI.list1 {list-style: none; color:white;}
 +
.blacktext {color:black}
 +
p, ul {
 +
    padding: 0;
 +
    margin: 0;
 +
}
 +
 +
</style>
<!-- Taskbar -->
<!-- Taskbar -->
<div id="taskbar">
<div id="taskbar">
Line 122: Line 205:
<img alt="Problem" src="/wiki/images/6/66/Darmstadt_green_Problem.jpg" width="100" height="30"></a>
<img alt="Problem" src="/wiki/images/6/66/Darmstadt_green_Problem.jpg" width="100" height="30"></a>
-
<a href="https://2013.igem.org/Team:TU_Darmstadt/solution">
 
-
<img alt="solution" src="/wiki/images/8/87/Darmstadt_green_Solution.jpg" width="100" height="30"></a>
 
<a href="https://2013.igem.org/Team:TU_Darmstadt/result">
<a href="https://2013.igem.org/Team:TU_Darmstadt/result">
Line 145: Line 226:
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook">
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook">
-
<img alt="team" src="/wiki/images/f/f3/Darmstadt_green_Labbook.jpg" width="90" height="30"></a>
+
<img alt="team" src="/wiki/images/4/4c/10._Labbook_(angewählt).jpg" width="90" height="30"></a>
-
</div>
+
</div><br>
-
<!-- In Fusion -->
+
 
 +
<br><br><br><br>
<center>
<center>
 +
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook">
 +
<font size="8" color="#F0F8FF" face="Arial regular">Lab book |</font>
 +
</a>
 +
<a href="https://2013.igem.org/Team:TU_Darmstadt/materials">
 +
<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font>
 +
</a>
 +
<a href="https://2013.igem.org/Team:TU_Darmstadt/protocols">
 +
<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font>
 +
</a>
 +
</center>
<br>
<br>
-
<br>
+
<!-- In Fusion -->
-
<br>
+
<center>
-
<br>
+
 
<h2><font size="6" color="#F0F8FF" face="Arial regular"> In Fusion </font></h2>
<h2><font size="6" color="#F0F8FF" face="Arial regular"> In Fusion </font></h2>
<div id="all">
<div id="all">
Line 162: Line 254:
<p text-aligne:left style="margin-left:50px; margin-right:50px">
<p text-aligne:left style="margin-left:50px; margin-right:50px">
<B> Materials<br></B>
<B> Materials<br></B>
-
5X In-Fusion HD Enzyme Premix
 
-
<br>pUC19 Control Vector,linearized (50 ng/μl)
 
-
<br>2 kb Control Insert (40 ng/μl)<br>
 
<br>
<br>
<B> Equipment<br></B>
<B> Equipment<br></B>
Line 171: Line 260:
<br>
<br>
<B> Chemicals & consumables <br></B>
<B> Chemicals & consumables <br></B>
 +
5X In-Fusion HD Enzyme Premix
 +
<br>pUC19 Control Vector,linearized (50 ng/μl)
 +
<br>2 kb Control Insert (40 ng/μl)<br>
<br>
<br>
<br>
<br>
<br>
<br>
<B> Procedure<br></B>
<B> Procedure<br></B>
-
1. <br>
+
1. Generating linearized vector<br>
-
2. <br>
+
2. Design gene-specific primers with 15 bp extensions homologous to vector end<br>
-
3. <br>
+
3. Amplify your gene of interest<br>
-
4. <br>
+
4. VolumeSet up the In-Fusion cloning reaction
 +
<p text-aligne:left style="margin-left:60px; margin-right:50px"><font size="3" color="#F0F8FF" face="Arial regular">
 +
    reaction mixture (20 µL total volume)<br><br>
 +
        1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)<br>
 +
        1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)<br>
 +
        1µl terminator (100 bp, c = 44 ng/µL, 44 ng)<br>
 +
        3,5µl TLO (2440 bp, c = 241,7 ng/µL, 780 ng)<br>
 +
        4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)<br>
 +
        3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)<br>
 +
        4µl 5x InFusion Pfu MasterMix<br>
 +
        1,5µl nucleasefree H2O<br></font></p>
 +
 
 +
<br><font size="3" color="#F0F8FF" face="Arial regular">
 +
<p text-aligne:left style="margin-left:50px; margin-right:50px">
 +
5.  Incubate cloning reaction for 15 min at 50°C<br>
 +
6.  Transform competent E. coli with the reaction mixture<br>
 +
 
<br>
<br>

Latest revision as of 00:36, 5 October 2013






Lab book | Materials | Protocols

In Fusion

Materials
Equipment
 In-Fusion® HD Cloning Kit
Micropipettes with sterile tips

Chemicals & consumables
5X In-Fusion HD Enzyme Premix
pUC19 Control Vector,linearized (50 ng/μl)
2 kb Control Insert (40 ng/μl)



Procedure
 1. Generating linearized vector
2. Design gene-specific primers with 15 bp extensions homologous to vector end
3. Amplify your gene of interest
4. VolumeSet up the In-Fusion cloning reaction

reaction mixture (20 µL total volume)

1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)
1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)
1µl terminator (100 bp, c = 44 ng/µL, 44 ng)
3,5µl TLO (2440 bp, c = 241,7 ng/µL, 780 ng)
4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)
3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)
4µl 5x InFusion Pfu MasterMix
1,5µl nucleasefree H2O


5. Incubate cloning reaction for 15 min at 50°C
6. Transform competent E. coli with the reaction mixture

Retrieved from "http://2013.igem.org/Team:TU_Darmstadt/protocols/InFusion"