Smolke:Protocols/Digest Screening

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Usage

  • When you cannot screen by colony PCR, mostly commonly because the insert is too small to generate a band shift or if the insert is of the same length as the cutout from the parent vector
  • Ideally should screen with restriction enzyme(s) that would cut both positive and negative colonies, but different number of times (e.g., single-cut for negative and double-cut for positive, or vice versa)

Procedure

  • Prepare plasmids using Maniatis method; phenol-chloroform extraction is optional if you do not plan to use the plasmid for anything other than screening
  • Set up 5-ul digestion reaction using the appropriate restriction enzymes
  • Should always include a negative control (most commonly the parent vector)
  • Should include a positive control if possible
  • Analyze on agarose gel; should include uncut plasmid for comparison
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