Team:Exeter/lab book/proto/4

From 2012.igem.org

(Difference between revisions)
(Created page with "{{Template:Team:Exeter/e-candi_banner}} <html xmlns="http://www.w3.org/1999/xhtml"> <head> <meta http-equiv="Content-Type" content="text/html; charset=utf-8" /> <title>Protocol ...")
Line 60: Line 60:
     <p><b>Digestion Protocol</b></p>
     <p><b>Digestion Protocol</b></p>
     <br>
     <br>
-
    <p>Upstream digest</p>
 
      
      
-
     <table cellpadding="10" cellspacing="10" style="font:Verdana; color:#1d1d1b; size:2">
+
     <table cellpadding="10" cellspacing="10">
     <tr>
     <tr>
-
       <td>Upstream Part Plasmid</td><td>500 ng</td>
+
       <td colspan="2" align="center"><u>Upstream digest</u></td><td></td><td colspan="2" align="center"><u>Downstream digest</u></td>
 +
    <tr>
 +
      <td>Upstream Part Plasmid</td><td>500 ng</td><td></td><td>Upstream Part Plasmid</td><td>500 ng</td>
     </tr>
     </tr>
     <tr>
     <tr>
-
       <td>EcoR1-HF</td><td>1 µl</td>
+
       <td>EcoR1-HF</td><td>1 µl</td><td></td><td>Xba1</td><td>1 µl</td>
     </tr>
     </tr>
     <tr>
     <tr>
-
       <td>Spe1</td><td>1 µl</td>
+
       <td>Spe1</td><td>1 µl</td><td></td><td>Pst1</td><td>1 µl</td>
     </tr>
     </tr>
     <tr>
     <tr>
-
       <td>10X NEBuffer2</td><td>5 µl</td>
+
       <td>10X NEBuffer2</td><td>5 µl</td><td></td><td>10X NEBuffer2</td><td>5 µl</td>
     </tr>
     </tr>
     <tr>
     <tr>
-
       <td>100X BSA</td><td>0.5 µl</td>
+
       <td>100X BSA</td><td>0.5 µl</td><td></td><td>100X BSA</td><td>0.5 µl</td>
     </tr>
     </tr>
     <tr>
     <tr>
-
       <td>H2O</td><td>to 50 µl</td>
+
       <td>H2O</td><td>to 50 µl</td><td></td><td>H2O</td><td>to 50 µl</td>
     </tr>
     </tr>
     </table>
     </table>
      
      
-
    <br>
 
-
   
 
-
    <p>Downstream digest</p>
 
-
    <table cellpadding="10" cellspacing="10">
 
-
    <tr>
 
-
      <td>Upstream Part Plasmid</td><td>500 ng</td>
 
-
    </tr>
 
-
    <tr>
 
-
      <td>Xba1</td><td>1 µl</td>
 
-
    </tr>
 
-
    <tr>
 
-
      <td>Pst1</td><td>1 µl</td>
 
-
    </tr>
 
-
    <tr>
 
-
      <td>10X NEBuffer2</td><td>5 µl</td>
 
-
    </tr>
 
-
    <tr>
 
-
      <td>100X BSA</td><td>0.5 µl</td>
 
-
    </tr>
 
-
    <tr>
 
-
      <td>H2O</td><td>to 50 µl</td>
 
-
    </tr>
 
-
    </table>
 
-
   
 
     <br>
     <br>
      
      
Line 142: Line 119:
     <p><b>Ligation Protocol</b></p>
     <p><b>Ligation Protocol</b></p>
     <p>Ligate the Upstream and Downstream Parts into the digested Destination Plasmid.</p>
     <p>Ligate the Upstream and Downstream Parts into the digested Destination Plasmid.</p>
-
   
+
    <br>
     <table cellpadding="10" cellspacing="10">
     <table cellpadding="10" cellspacing="10">
     <tr>
     <tr>

Revision as of 15:13, 19 September 2012

Protocol 4

New England BioLabs Biobrick Assembly Protocol

Digestion Protocol


Upstream digestDownstream digest
Upstream Part Plasmid500 ngUpstream Part Plasmid500 ng
EcoR1-HF1 µlXba11 µl
Spe11 µlPst11 µl
10X NEBuffer25 µl10X NEBuffer25 µl
100X BSA0.5 µl100X BSA0.5 µl
H2Oto 50 µlH2Oto 50 µl

Digest the Destination Plasmid with EcoR1-HF and Pst1: The Destination Plasmid DNA should either be prepared with PCR or contain a toxic gene (e.g. ccdb, sacB) in the cloning site to avoid the need for gel purification. The Destination Plasmid should also have a different antibiotic resistance marker from both the plasmid containing the Upstream Part and the plasmid containing the Downstream Part to avoid the need to purify the Upstream and Downstream Parts.


Destination Plasmid DNA500 ng
EcoR1-HF1 µl
Pst11 µl
10X NEBuffer25 µl
100X BSA0.5 µl
H2Oto 50 µl

Incubate all three restriction digest reactions at 37 °C for 10 minutes and then heat inactivate at 80 °C for 20 minutes.


Ligation Protocol

Ligate the Upstream and Downstream Parts into the digested Destination Plasmid.


Upstream Part digestion2 µl
Downstream Part digestion2 µl
Destination Plasmid digestion2 µl
10X T4 DNA ligase Buffer2 µl
T4 DNA ligase1 µl
H2O11 µl

Incubate at room temperature for 10 minutes and then heat inactivate at 80 °C for 20 minutes.

Retrieved from "http://2012.igem.org/Team:Exeter/lab_book/proto/4"