Mike Barnkob:Protocols/Bacterial/Diagnostic Restriction Digest

From OpenWetWare
Jump to: navigation, search

Front page

Diagnostic Restriction Digest

Diagnostic restriction digest to verify plasmid identity.

Reagents

  • Restriction enzymes
  • DNA ladder
  • Gel

Protocol

  1. 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.
  2. 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:
      1. Uncut samples: add 25 μL
      2. Without DNA: add 26 μL
      3. With 1 RE: 25 μL
      4. With 2 RE: 24 μL
    • Mix gently
  3. Incubate tube in 37°C water bath for 1 hour.
  4. Make gel when digestion is running.
  5. Inactivate reaction by incubating tube on 70°C heat-block for 15 min.
  6. Run reaction on gel electrophoresis: 40 min at 100 V with 5 μL Loading Buffer.

References