Smolke:Protocols/Digest Screening

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