RestrictionDigestsofHighMolecularWeightYeastDNA

Purpose:

• To perform restriction digests of YACs for mapping using rare cutting enzymes or more conventional restriction endonuclease digestion.

Time required:

• 2-4 hours

Special Reagents:

• dependent upon enzymes desired

Procedure:

1. Using wide-bore pipette, aliquot 100 ng of liquid DNA into eppendorf tube. Add 2.5 祃 10X restriction buffer, 1-2 ul RNase (5 mg/ml stock), 250-500 ng BSA, then dH2O up to 25 祃 volume. Incubate at appropriate temperature for 2-4 hours.

2. Stop the reaction by adding 3 祃 of 10X glycerol stop mix; mix in tube gently, then microfuge for 15 seconds (rather than pipette up and down, which may shear the DNA).