Richard Lab:Restriction Digest protocol

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Solutions/reagents:

• prepared DNA
  
  (from Miniprep, PCR or Gel Extraction)
• restriction endonucleases
• 10X restriction endonuclease buffer
• BSA
  
  (optional)
• phosphatase
• phosphatase buffer
• distilled water

Equipment:

• Incubator
• Sterile 1.5-ml microcentrifuge tubes

Steps:

1. Quickly vortex all ingredients (Buffer, BSA, DNA, Enzymes) before beginning.
   
2. Measure out prepared DNA into sterile 1.5-ml microcentrifuge tube (1).
   Add 4 volumes distilled water.
   Use the following table as a checklist for preparing the reaction in sterile 1.5-ml microcentrifuge tube (2):
   
   

   	10X restriction endonuclease buffer	BSA	DNA solution	restriction endonucleases
   Restriction Digestion	5 µl	1 µl	40 µl	4 µl

3. Incubate at 37°C for at least 1 hr.
   Use a water bath for incubation.
   
4. Perform enzyme inactivation by storing at 75°C for 15 mins.
   
5. If digesting vector:
   Measure out 1 µl of phosphatase into sterile 1.5-ml microcentrifuge tube (2).
   Add 5 µl of phosphatase buffer.
   Incubate at 37°C for at least 45 mins.
   Perform enzyme inactivation by storing at 75°C for 15 mins.
   
6. Store at -20°C.
   

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 2 hrs, 15 mins