Team:TU Darmstadt/protocols/Colony PCR

From 2013.igem.org

(Difference between revisions)
 
(3 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;
 +
}
 +
 +
 +
 +
 +
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;}
LI.list1 {list-style: none; color:white;}
Line 117: Line 195:
     margin: 0;
     margin: 0;
}
}
 +
</style>
</style>
 +
 +
<body>
 +
 +
<center>
 +
<!-- central main menu -->
 +
 +
<br>
 +
<br>
<!-- Taskbar -->
<!-- Taskbar -->
Line 127: Line 214:
<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 150: Line 235:
<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><br><br><br>
 +
<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>
<!-- Colony PCR -->
<!-- Colony PCR -->
<center>
<center>
<br>
<br>
-
<br>
+
 
-
<br>
+
-
<br>
+
<h2><font size="6" color="#F0F8FF" face="Arial regular"> Colony PCR </font></h2>
<h2><font size="6" color="#F0F8FF" face="Arial regular"> Colony PCR </font></h2>
<div id="all">
<div id="all">

Latest revision as of 00:31, 5 October 2013







Lab book | Materials | Protocols

Colony PCR

Materials


Equipment

  • - PCR machine


Chemicals & consumables

  • - Sterile Eppendorf Tubes
  • - LB-agar plate with appropriate antibiotic
  • - Primers (usually VF2 and VR)
  • - Sterile pipet tips


Procedure


The colony PCR is a modified PCR programm employed to verifiy transformation success by amplifiying the insert or the vector construct used for transformation. This is necessary due to the fact, that a transformation with the empty vector may lead to antibiotic resistance.

  1. Pick one colony with a sterile tip and suspend in 10 µL of ddH2O.
  2. Inoculate tip with colony into tube. Pipet up and down to ensure all cells are transferred to tube.
  3. Start the PCR using the following programm and 1X mix.
  4. Run a gel to determine the product length (don't forget the positiv control).


# Temperature Time
1 95 °C 00:05:00
2 95 °C 00:00:20
3 62 °C 00:00:30
4 68 °C 00:02:00
5 GO TO 2 REPEAT 30x
6 68 °C 00:05:00
7 4 °C HOLD


Mixtures


1X Reaction Mixture

  • - 2 µL of 10x Thermopol Reaction Buffer
  • - 0,4 µL of dNTPs (10 mM each)
  • - 0,3 µL of Taq DNA Polymerase
  • - VF2 (10 pmol)
  • - VR (10 pmol)
  • - 0,6 µL of DMSO
  • - 1 µL of colony suspension
  • - ddH2O to 20 µL