Categorized: Bacterial Genetics
- Isolate chromosomal DNA by using the CTAB protocols in current Pr M R.
- Make a CsCl preparation of pLAFr3 (or other appropriate cosmid).
- Cut pLAFr with BamHI phenol ext. + ETOH preparation.
- 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.
- Scale up the conditions (all of the conditions!!) to obtain 200 – 500 µg of cut DNA.
- Prepare a 10 – 40% sucrose gradient as per Maniatis (pp. 2.85 – 2.87).
- Pool fractions containing 20 kb fragments and, after adding 3 vol. of sterile dH2O, prep. the DNA.
- Set up ligations (Maniatis p. 3.29) to maximize the formation of concatomers.
- 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.
- Check a randomly selected group of transformants for the presence of inserts by restriction analysis.