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

Revision as of 08:53, 27 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.
- For each plasmid, prepare lane without any restriction enzymes and one without any DNA.
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 20 μL with dH20.
- Mix gently
Incubate tube in 37°C water bath for 1 hour.
Make gel when digestion is running.
Inactivate reaction by incubating tube on 70°C heat-block for 15 min.
Run reaction on gel electrophoresis: 35 min at 100 V with 5 μL Loading Buffer.

@@ Line 22: / Line 22: @@
 #* 3 μL of 10x reaction buffer
 #* 3 μL of BSA if recommended
-#* Bring total volume to 30 μL with dH20.
+#* Bring total volume to 20 μL with dH20.
 #* Mix gently
 # Incubate tube in 37°C water bath for 1 hour.
 # Make gel when digestion is running.
 # Inactivate reaction by incubating tube on 70°C heat-block for 15 min.
-# Run reaction on gel electrophoresis.
+# Run reaction on gel electrophoresis: 35 min at 100 V with 5 μL Loading Buffer.
 ===References===
 * http://www.addgene.org/recipient-instructions/diagnostic-digest/