Method: Restriction Digests of High Molecular Weight Yeast DNA

Jim Howe

May 5, 1990

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 µl 10X restriction buffer, 1-2 ul RNase (5 mg/ml stock), 250-500 ng BSA, then dH2O up to 25 µl volume. Incubate at appropriate temperature for 2-4 hours.
2. Stop the reaction by adding 3 µl 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).