Cosmid Library

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.