Mike Barnkob:Protocols/Bacterial/Diagnostic Restriction Digest: Difference between revisions

Revision as of 07:59, 23 January 2015

Diagnostic restriction digest to verify plasmid identity.

Select restriction enzymes and correct buffer to digest plasmid
- For Addgene plasmids, use their suggested enzymes and compare
- Use NEBs Double Digest Finder to find the correct reaction buffer and if BSA is recommended or not.
Prepare reaction mix in 1.5 mL Eppendort tube:
- 2 μg DNA
- 1 μL of each restriction enzyme
  Note: Keep all enzymes on ice or ice-block all the time!
- 3 μL of 10x reaction buffer
- 3 μL of BSA if recommended
- Bring total volume to 30 μL with dH20.
- Mix gently
Incubate tube in 37°C water bath for 1 hour.
Inactivate reaction by incubating tube on 70°C heat-block for 15 min.
Run reaction on gel electrophoresis.

@@ Line 9: / Line 9: @@
 * Restriction enzymes
 * DNA ladder
+* Gel
 ===Protocol===
-#
+# Select restriction enzymes and correct buffer to digest plasmid
+#* For Addgene plasmids, use their suggested enzymes and compare
+#* Use NEBs [https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder Double Digest Finder] to find the correct reaction buffer and if BSA is recommended or not.
+# Prepare reaction mix in 1.5 mL Eppendort tube:
+#* 2 μg DNA
+#* 1 μL of each restriction enzyme <br />'''Note:''' Keep all enzymes on ice or ice-block all the time!
+#* 3 μL of 10x reaction buffer
+#* 3 μL of BSA if recommended
+#* Bring total volume to 30 μL with dH20.
+#* Mix gently
+# Incubate tube in 37°C water bath for 1 hour.
+# Inactivate reaction by incubating tube on 70°C heat-block for 15 min.
+# Run reaction on gel electrophoresis.
 ===References===
 * http://www.addgene.org/recipient-instructions/diagnostic-digest/