Mike Barnkob:Protocols/Bacterial/Diagnostic Restriction Digest
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Diagnostic Restriction Digest
Diagnostic restriction digest to verify plasmid identity.
- Restriction enzymes
- DNA ladder
- 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 control samples: one without any restriction enzymes (uncut) and one without any DNA.
- Prepare reaction mix in 1.5 mL Eppendort tube:
- 1-2 μg of 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. Usually around:
- Uncut samples: add 25 μL
- Without DNA: add 26 μL
- With 1 RE: 25 μL
- With 2 RE: 24 μL
- 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: 40 min at 100 V with 5 μL Loading Buffer.