• Restriction Digests of High Molecular Weight Yeast DNA

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Donis-Keller Lab Manual, Department of Genetics in Washington University School of Medicine http://hg.wustl.edu/hdk_lab_manual/yeast/yeast5.html 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).

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