Construct phage library

My phage library usually has a backbone based on the fdg3p0ss phage plasmids, which has only one pair of disulfide bridge embedded deep in the phage PIII protein. Fdg3p0ss was further modified by inserting two SfiI sites to allow the insertion of the peptide library. One SfiI site is exactly after the pIII protein secretion signal, and another one is in the middle of pIII protein. The procedure I am using to prepare the phage libraries is described as following:

  1. Design primers as above to generate the insert flanked by two SfiI sites.
  2. Use the insert gene as template to PCR clone the library insert, and agrose gel purify the insert.
  3. SfiI digest the insert at 50°C over night.
  4. Use QIAGEN kit cleanup the digested insert.
  5. Digest the phage vector at 50°C over night, and purify the vector in agrose gel.
  6. Insert:vector=3:1 molar ratio was ligated with T4 ligase at 16°C for one day.
  7. Cleanup the ligation mixture and elute the DNA with mQ water.
  8. Transform the library DNA into TG1 cells with electroporation.
  9. Incubate the transformation mixture at 37°C for 1hour without shaking.
  10. Titer the TG1 cells with chloramphenicol resistance to determine the diversity of the library. The size of the library should be larger and 5E8.
  11. After being incubated at 37°C ON, the phage library is scriped from the agar plate and mixed with glycerol and flash freezed and stored at -80°C with 1mL in each stock tube.