西亚试剂：：Cosmid Library

发布时间：2025-10-05

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.

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