Team:Groningen/protocols/Ligation

From 2013.igem.org

(Difference between revisions)
Line 37: Line 37:
<table border = "1">
<table border = "1">
<tr><td>Component</td>
<tr><td>Component</td>
-
<td>30&#956;l reaction</td>
+
<td>20&#956;l reaction</td>
<td>Final concentration</td>
<td>Final concentration</td>
</tr>
</tr>
<tr><td>H<sub>2</sub>O</td>
<tr><td>H<sub>2</sub>O</td>
-
<td>up to 30&#956;l</td>
+
<td>up to 20&#956;l</td>
<td></td>
<td></td>
</tr>
</tr>
-
<tr><td>Buffer 10x</td>
+
<tr><td>10x Ligation buffer</td>
-
<td>3&#956;l</td>
+
<td>2&#956;l</td>
<td>1x</td>
<td>1x</td>
</tr>
</tr>
-
<tr><td>DNA</td>
+
<tr><td>Vector</td>
-
<td>4&#956;l</td>
+
<td>1&#956;l</td>
-
<td>up to 1&#956;g</td>
+
<td>20-100&#956;g</td>
</tr>
</tr>
-
<tr><td>Enzyme/s</td>
+
<tr><td>Insert</td>
-
<td>0.5&#956;l</td>
+
<td>1&#956;l</td>
-
<td></td>
+
<td>1:1 molar ratio</td>
 +
</tr>
 +
<tr><td>T4 DNA Ligase</td>
 +
<td>0.2&#956;l</td>
 +
<td>5U/&#956;l</td>
</tr>
</tr>
</table>
</table>

Revision as of 10:05, 26 July 2013

Ligation

Materials:

  • H2O
  • 1.5ml tubes
  • Ligation buffer
  • Ligase
  • Insert
  • Vector

Reaction:

Component 20μl reaction Final concentration
H2O up to 20μl
10x Ligation buffer 2μl 1x
Vector 1μl 20-100μg
Insert 1μl 1:1 molar ratio
T4 DNA Ligase 0.2μl 5U/μl

All the reagents are added following the order listed in the table above.
After the reaction is ready mix the content of the tube and spin it down.
The tubes are incubated for 1h at 37°C.

Reference: http://www.thermoscientificbio.com/uploadedFiles/Resources/fast-digestion-dna.pdf