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
|
