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Extra info for An Introduction to the Philosophy of Mind
The YACt2 vector was digested with MluI (compatible with BssHII on ligation) and BarnHI, phosphatased and ligated to the genomic DNA. After removing some of the nonrecombmant vector on an agarose gel, the ligation mix was transformed into the Ura- yeast strain AB 1380. The 4p telomere was identified from the background of vector and nontelomeric clones by hybridtzation with a 4p specific probe. 7. Novozym 234 (Novobiolabs, Denmark), Zymolyase 20T (ICN Biochemicals, High Wycombe, UK). 8. Lyticase (Sigma).
2. Prepare a PCR premix suffictent for each set of 16 secondary pools, plus the positive primary pool and a genomic and To iE control. 1. 3. Identify the positive secondary pools. Each pool represents the contents of a single mrcrotiter plate in the poolmg systemjust described (see Note 24). 3. Preparation of “Rows and Columns” PCR Preparation of rows and columns PCR pools can be avoided by the use of hybridization to nylon filters (see Note 1). 1. Thaw YAC library (working) mtcrotiter plate correspondmg to each positive secondary pool.
Hybridize the filters as described. 8. Use the second plate for archiving the appropriate clones (see Note 19). 2. 1. 5~mL microcentrifuge tubes. For rapid throughput of STSs, pools are stored in 96-well microtiter plates enabling the use of multichannel pipets to set up the reactions in PCR microtiter plates and to load the agarose gels (see Note 16, Chapter 3). 1. 7 yL To lE. Vortex to mix. 2. 5~uL Eppendorf tube approx 5 mm below the lip of the tube. 3. Place a 3 uL droplet of T,, ,E and 3 uL 25 ng/uL genomtc DNA onto the edge of the negative and positive control tubes.