UCL London/Protocol/Ligation

(Difference between revisions)

Latest revision as of 11:09, 18 October 2009

Materials:

Plasmid DNA: 15 µL
Restriction Enzymes (depend on the requirements of the parts)
- EcoRI: 1 µL
- PstI: 1 µL
- SpeI: 1 µL
- XbaI: 1 µL
10× NE buffer: 1.5 µL/1 µL (lid)
BSA: 1 µL (lid)

Method:

Digest the DNA as in Restriction Enzyme Digestion Protocol.
Heat inactive the restriction enzymes.
Run a diagnostic gel before the ligation.
After the gel is confirmed, add 1 µL quick ligase, 10 µL ligation buffer, 3 µL backbone and 6 µL inserts (or other volumes depend on the concentration of DNA) into an eppendorf.
Leave for 5 minutes (or up to 30 minutes) at room temperature (25°C).
Transform the ligated DNA onto competent cells as described in Transformation, method 2 step 4 to 10.

UCL London IGEM Wiki 2009

@@ Line 3: / Line 3: @@
 <div id="ucla1">
+: '''''Materials:'''''
+* Plasmid DNA: 15 µL
+* Restriction Enzymes (depend on the requirements of the parts)
+** EcoRI: 1 µL
+** PstI: 1 µL
+** SpeI: 1 µL
+** XbaI: 1 µL
+* 10× NE buffer: 1.5 µL/1 µL (lid)
+* BSA: 1 µL (lid)
+* Quick Ligase
+* 2× Quick Ligation Buffer
+: '''''Method:'''''
+# Digest the DNA as in [[UCL_London/Protocol/Restriction_Enzyme_Digestion|Restriction Enzyme Digestion Protocol]].
+# Heat inactive the restriction enzymes.
+# Run a diagnostic gel before the ligation.
+# After the gel is confirmed, add 1 µL quick ligase, 10 µL ligation buffer, 3 µL backbone and 6 µL inserts (or other volumes depend on the concentration of DNA) into an eppendorf.
+# Leave for 5 minutes (or up to 30 minutes) at room temperature (25°C).
+# Transform the ligated DNA onto competent cells as described in [[UCL_London/Protocol/Transformation| Transformation]], method 2 step 4 to 10.
 </div>
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