Restriction Digests of YACs in Agarose Plugs

Purpose:

To perform restriction enzyme digests on intact, high molecular weight YAC DNA, for use in pulsed-field mapping experiments.

Time required:

2 days

Special Reagants:

• Rare cutting enzymes need to be chosen well in advance so that they can be ordered and received by the time of the experiment.

Procedure:

Day 1

1. Transfer YAC plugs which have been stored in 50 mM EDTA to a 6 well tissue culture plate. Add 3-5 ml of TE (pH 7.6) to the wells containing YAC plugs and incubate at room temperature for 30 minutes. Transfer plugs to an equal volume of fresh TE and continue to incubate for 30 minutes.

2. Cut plugs into rectangular pieces 1/8-1/4 the size of the whole plug (size that will fill a well on the CHEF DR apparatus), and transfer to eppendorf tubes. Add 250 祃 of 1X restriction buffer, then incubate at room temperature for 30 minutes. Aspirate buffer, and replace with fresh 1X buffer for an additional 30 minutes at room temperature. Aspirate buffer, then replace with 1X buffer containing 100 礸/ml BSA. Add 30-50 units of the appropriate restriction enzyme, then incubate overnight.

Day 2

1. Aspirate liquid. Add 250 祃 of 0.5X TBE and incubate at room temperature with gentle shaking for 1 hour. Place plugs in the wells of a prepared CHEF gel, and seal with 1% low-melt agarose. Run CHEF gel under conditions ideal for the separation of the expected size fragments for these enzymes.

References:

Smith, C.L., Lawrance, S.K., Gillespie, G.A., Cantor, C.R., Weissman, S.W., and F. S. Collins. (1987). "Strategies for mapping and cloning macroregions of mammalian genomes." In Methods in Enzymology, Wu et al eds., vol. 151.

Sambrook, J., Fritsch, E.F., and T. Maniatis.(1989) Molecular Cloning, A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press, p.6.57.