Anyone has a good protocol for transforming cosmid into E.Coli? Mine is about 40-50kb.

Thanks.

COHscientist:

You can find a great overview of working with high-capacity vectors including using cosmid transformation to generate genomic libraries in:

Sambrook and Russell. Molecular Cloning, A Laboratory Manual - Volume 1. Cold Springs Harbor Laboratory Press, New York, NY. 2001, 3rd Edition. Chapter 4, p 4.1 - 4.92.

The first four protocols in this chapter may be helpful to you:

Protocol 1: Construction of Genomic DNA Libraries in Cosmid Vectors, 4.11 - 4.23

Protocol 2: Screening an Unamplified Cosmid Library by Hybridization: Plating the Library onto Filters, 4.24 - 4.27

Protocol 3: Amplification and Storage of a Cosmid Library: Amplification in Liquid Culture, 4.28 - 4.30

Protocol 4: Amplification and Storage of a Cosmid Library: Amplification on Filters, 4.31 - 4.34


Each of the above protocols deal with using bacteriophage lambda, however if you prefer to work with bacteriophage P1, you may want to use these protocols:

Protocol 5: Working with Bacteriophage P1 and Its Cloning Systems, 4.35 - 4.45

Protocol 6: Transferring Bacteriophage P1 Clones between E. coli Hosts, 4.46 - 4.47


Other protocols that may interest you in this chapter are:

Protocol 7: Working with Bacterial Artificial Chromosomes, 4.48 - 4.52

Protocol 8: Isolation of BAC DNA from Small-Scale Cultures, 4.53 - 4.54

Protocol 9: Isolation of BAC DNA from Large-Scale Cultures, 4.55 - 4.57

Protocol 10: Working with Yeast Artificial Chromosomes, 4.58 - 4.66

Protocol 11: Growth of S. cerevisiae and Preparation of DNA, 4.67 - 4.69

Protocol 12: Small-Scale Preparations of Yeast DNA, 4.70 - 4.71

Protocol 13: Analyzing Yeast Colonies by PCR, 4.72 - 4.73

Protocol 14: Isolating the Ends of Genomic DNA Fragments Cloned in High Capacity Vectors: Vectorette Polymerase Chain Reaction, 4.74 - 4.81



Please note that the 3-volume set of Sambrook and Russell Molecular Cloning Manuals can be a bit expensive, so it may be of benefit to you to see if someone in your lab already owns these.

Good Luck,

Tony Rook


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