Richard Lab:Restriction Digest protocol
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Solutions/reagents:
- <a name="prepared DNA">prepared DNA
<tab>(from Miniprep, PCR or Gel Extraction)</a> - restriction endonucleases
- 10X restriction endonuclease buffer
- <a name="BSA">BSA
<tab>(optional)</a> - phosphatase
- phosphatase buffer
- distilled water
Equipment:
- Incubator
- Sterile 1.5-ml microcentrifuge tubes
Steps:
- Quickly vortex all ingredients (Buffer, BSA, DNA, Enzymes) before beginning.
- Measure out <a href="#prepared DNA" >prepared DNA</a> 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):
<thead></thead><tbody></body>10X restriction endonuclease buffer BSA DNA solution restriction endonucleases Restriction Digestion 5 µl 1 µl 40 µl 4 µl - Incubate at 37°C for at least 1 hr.
Use a water bath for incubation. - Perform enzyme inactivation by storing at 75°C for 15 mins.
- 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. - Store at -20°C.
TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 2 hrs, 15 mins
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