Richard Lab:Restriction Digest protocol

Solutions/reagents:  prepared DNA  (from Miniprep, PCR or Gel Extraction) restriction endonucleases10X restriction endonuclease buffer BSA  (optional) phosphatasephosphatase bufferdistilled water</li></ul> Equipment: Incubator</li>Sterile 1.5-ml microcentrifuge tubes</li></ul> Steps: <ol>  Quickly vortex all ingredients (Buffer, BSA, DNA, Enzymes) before beginning. </li> Measure out <a href="#prepared DNA" ><font color=#357EC7>prepared DNA </a> into sterile 1.5-ml microcentrifuge tube (1). Add <font color=#357EC7>4  volumes <font color=#357EC7>distilled water. Use the following table as a checklist for preparing the reaction in sterile 1.5-ml microcentrifuge tube (2): </li>  Incubate at <font color=#357EC7>37°C  for at least <font color=#357EC7>1 hr . <font color = "#800517">Use a water bath for incubation. </li> Perform enzyme inactivation by storing at <font color=#357EC7>75°C  for <font color=#357EC7>15 mins . </li>  If digesting vector:  Measure out <font color=#357EC7>1 µl  of <font color=#357EC7>phosphatase into sterile 1.5-ml microcentrifuge tube (2). Add <font color=#357EC7>5 µl  of <font color=#357EC7>phosphatase buffer. Incubate at <font color=#357EC7>37°C  for at least <font color=#357EC7>45 mins . Perform enzyme inactivation by storing at <font color=#357EC7>75°C  for <font color=#357EC7>15 mins . </li> Store at <font color=#357EC7>-20°C . </li> </ol> TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 2 hrs, 15 mins