Endy:Library-based construction/Inverter library protocol

We should be fleshing this out as we work through it a few times

Generate inverter library via mutagenic PCR

• 50 ul PCR reaction using Stratagene's GeneMorph® II Random Mutagenesis kit
• PCR cleanup and elute with 30 ul

Digest the library and ligate into SP1.0

• Example:
  • Perform a 50 ul restriction digest using all of the PCR reaction
  • Separate the insert out on a gel and perform gel extraction
  • Elute the entire reaction into a single 30 ul volume (reuse on multiple columns if necessary)
  • Perform two 20-ul ligations with 15 ul of the digest in each and 1 ul of the cut SP1.0 vector
    • Can also save some of the ligation and use less, probably don't need this much

Transform into electrocompetent CW2553/pJat8

1. Grow for one hour without antibiotic (1ml), then move into a higher volume (50ml) of selective media
   • plate a portion on selecive media in order to determine the libary size.
2. Store 5 glycerols of the libary from the overnight culture
3. Start experimental cultures with no induction and high induction
4. Sort cells with LOW in/HIGH out and HIGH in/LOW out into sub-libraries
   • Be sure to also collect analytical samples of the populations after sorting, so that we have data of what the libraries looked like.
5. Grow up the sub-libraries under the reverse condition (and the same condition)
   • The reason for growing under the same conditions is to see how effective the sorting was (control, basically).
6. Conduct second round of sorting to create sub-libraries that we're tested under both HIGH input and LOW input conditions
7. Grow up library and store 5 glycerols, as well as plate to get individual mutants
8. Test individual mutants, if necesary can repeat the rounds of screening to tighten up the library