Team:TU Darmstadt/protocols/Chemically competent cells

From 2013.igem.org

(Difference between revisions)
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 128: Line 206:
<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 151: Line 227:
<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>
 +
<!-- Chemically competent cells -->
<!-- Chemically competent cells -->

Revision as of 23:41, 4 October 2013





Chemically competent cells

Materials


Equipment

  • - -80°C freezer
  • - Incubation shaker
  • - Centrifuge (cooling cababilities required!)
  • - photometer
  • - Ice water bath


Chemicals & consumables

  • - Ice and/or liquid nitrogen
  • - Falcon tubes
  • - dYT Medium (50 ml p.c.)
  • - ice cold 100mM CaCl2
  • - Glycerin


Procedure


The transformation of E. coli with plasmid DNA via heatshock transformation requires chemically competent cells.

  1. Inoculate 2 mL of LB-Media with an E. coli colony and incubate at 37 °C overnight.
  2. Inoculate 200 mL LB with the preculture.
  3. Incubate at 37°C and 150 rpm until an OD600 of 0.4-0.6 is reached.
  4. Incubate cells on ice for 15 min.
  5. Centrifuge the culture at 4°C and 3000 x g for 10 min (the following steps are carried out on ice).
  6. Resuspend cell pellet in 10mL ice cold 100 mM CaCl2 (Do not vortex!).
  7. Incubate on ice for 1 hour.
  8. Centrifuge the culture at 4°C and 3000 x g for 10 min.
  9. Resuspend cell pellet in 10mL ice cold 100 mM CaCl2.
  10. Incubate on ice for 1 hour.
  11. Centrifuge the culture at 4°C and 3000 x g for 5 min.
  12. Resuspend cell pellet in 2mL ice cold 100 mM CaCl2 and 15 % (v/v) glycerine.
  13. Incubate on ice for 30 min.
  14. Aliquot the cells à 100µ.
  15. Store at -80°C.


Mixtures


CaCl2-Solution

  • - 5.55 g CaCl2
  • - add ddH2O to 1 L
  • - sterilize by autoclaving


Cryo solution

  • - 0.278 g CaCl2
  • - 10 ml glycerin
  • - Add ddH2O to 50 ml
  • - Sterilize by autoclave

References

  1. Mandel, M. and Higa, A.: Calcium-dependent bacteriophage DNA infection. J Mol Biol, 1970, 53, 159-162

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