|
| Size of Yeast Genome: |
1.5 X 107 bp |
| Size of average YAC: |
3.0 X 105 bp |
| Size of average Inserts: |
2.0 X104 bp |
| # Inserts to cover Genome: |
750 |
| Number of plaques to screen to cover |
|
| the YAC genome 3-5 times: |
2250-3750 |
| Number of plates needed (375 plaques each): |
10 |
It is important to remember that you have cloned the entire yeast genome as well as the YAC into the phage, and thus need to devise a strategy of recovering human containing recombinants.
Prepare filters from each plate (as described in phage methods section for plaque lifts), and hybridize with total human DNA to identify the human clones by the presence of highly repetitive sequences, e. g., Alu sequences. Hybridization of the plaque lifts to total yeast DNA identifies yeast clones with yeast repetitive DNA. Using probes you know are located on the YAC to screen the YAC subclones will identify clones containing those sequences. The ends of the human insert can be identified by hybridizing the plaque lifts to pBR322 to detect the subclones containing portions of the vector arms (see the procedure for the identification of end clones in YAC subclone libraries).
Solutions:
References:
Sambrook, J., Fritsch, E.F., and T. Maniatis.(1989) Molecular Cloning, A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press. p. 2.32, 2.82-2.98.
Instruction manual to Stratagene Undigested EMBL3 Cloning Kit, 1989.
Instruction manual to Gigapack II XL Packaging Extract protocol, 1989.
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