Cosmid Library

1. Isolate chromosomal DNA by using the CTAB protocols in current Pr M R.
   
2. Make a CsCl preparation of pLAFr3 (or other appropriate cosmid).
   
3. Cut pLAFr with BamHI phenol ext. ETOH preparation.
   
4. Partially digest a sample of chromosomal DNA as per Maniatis protocol (pp. 9.24 - 9.28) in order to maximize production of 20 kb plasmid.
   
5. Scale up the conditions (all of the conditions!! to obtain 200 - 500 ?g of cut DNA.
   
6. Prepare a 10 - 40% sucrose gradient as per Maniatis (pp. 2.85 - 2.87).
   
7. Pool fractions containing 20 kb fragments and, after adding 3 vol. of sterile dH₂O, prep. the DNA.
   
8. Set up ligations (Maniatis p. 3.29) to maximize the formation of concatomers.
   
9. Package via "Packagene" protocol. Plate the infected bacteria on LB Tet. plates. Remember: if you are using pLAFr, you are looking for colonies, NOT PLAQUES, since it is a cosmid.
   
10. Check a randomly selected group of transformants for the presence of inserts by restriction analysis.