METHOD:

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 - 500μg 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 dH2 O, 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.