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  • Detection using the Phototope-Star Detection Kit

    Overview

    The following protocol will allow detection of biotinylated DNA on Southern and Northern Blots, dot blots, and DNA sequencing membranes (2,5,6).  Detection of plaque lifts and colony hybridizations can be obtained using standard protocols.  The kit is not yet recommended for in situ hybridization.

    Note: In all steps during the detection procedures, it is important that the membrane is evenly covered with solution and that it floats freely in solution.  If a hybridization bag is used, it must be free of trapped air to ensure maximum contact between the reagent and the membrane.  Examples of the detection volumes necessary for a 10 cm x 10 cm membrane sealed in a hybridization bag are given.  Volumes required for detections done in a container, or bag that is considerably larger than the membrane may be greater.  Be sure that the membrane stays completely wetted.

    1. Wash Step - Detection Solution A

      Add to the hybridization bag, 0.1 ml of Detection Solution A per cm2 of membrane.

      Incubate for 5 minutes at room temperature with moderate shaking. 

      Drain the solution before the next step.

      Sample calculation
      100 cm2 x 0.1 ml/cm2 = 10 ml

    2. Streptavidin incubation

      Determine the necessary volume of diluted streptavidin based on using 0.05 ml of diluted streptavidin per cm2 of membrane.

      Prepare this volume by diluting the streptavidin stock solution 1:1000 with Detection Solution A.  Incubate for 5 minutes at room temperature with moderate shaking.

      Drain the solution before the next step.

      Sample calculation
      100 cm2 x 0.05 ml/cm2 = 5 ml.

      Dilute 5 μl of streptavidin stock solution to 5 ml with Detection Solution A.

    3. Wash 2 times - Detection Solution B

      Use 0.5 ml Detection Solution B per cm2 of membrane.

      Wash twice, 5 minutes each, at room temperature with moderate shaking.

      Drain and discard the solution after each wash.

      Sample calculation
      100 cm2 x 0.5 ml/cm2 = 50 ml each.

    4. Biotinylated Alkaline Phosphatase incubation

      Determine the necessary volume of diluted phosphatase based on using 0.05 ml of diluted biotinylated alkaline phosphatase per cm2 of membrane.

      Prepare this volume by diluting the biotinylated alkaline phosphatase stock solution 1:1000 with Detection Solution A.  Incubate for 5 minutes at room temperature with moderate shaking.

      Drain the solution before the next step.

      Sample calculation
      100 cm2 x 0.05 ml/cm2 = 50 ml

      Dilute 5 μl biotinylated alkaline phosphatase stock solution to 5 ml with Detection Solution A.

    5. Wash 1 time - Detection Solution A

      Use 0.5 ml Detection Solution A per cm2 of membrane.

      Wash once for 5 minutes at room temperature with moderate shaking.

      Drain and discard the solution.

      Sample calculation
      100 cm2 x 0.5 ml/cm2 = 50 ml

    6. Wash 2 times - Detection Solution C

      Use 0.5 ml of 1X Detection Solution C per cm2 of membrane.

      Wash twice, 5 minutes each, at room temperature with moderate shaking.

      Drain and discard the solution after each wash.

      Sample calculation
      100 cm2 x 0.5 ml/cm2 = 50 ml each

    7. Detecting the DNA

      The CDP-Star Reagent is supplied as a 25 mM stock solution and should ony be diluted immediately before use.  CDP-Star Dilution Buffer is supplied as a 25X stock solution.

      CDP-Star Reagent stock solution can be diluted from 1:100 to 1:500* (CDP-Star manufacturer recommends 1:100 dilution for all applications) depending on its intended use.  For experiments requiring maximum signal intensity and longetivity, dilute 1:100 with 1X CDP-Star Dilution Buffer.  For maximum volume in experiments where sensitivity is not a problem, dilute up to 1:500 with 1X CDP-Star Dilution Buffer.  Sufficient reagents are provided for detection on 4,000 cm2 of membrane using the maximum sensitivity protocol or 20,000 cm2 of membrane using the maximum volume protocol. 

      A. Dilute the CDP-Star (25X) Dilution Buffer with Milli-Q™ water to a 1X concentration. 1X CDP-Star Dilution Buffer is stable at room temperature; therefore, large quantities can be prepared and stored for extended periods of time.

      B. Determine the amount of diluted CDP-Star Reagent needed and prepare this quantity immediately before use. Use 0.025 ml of diluted CDP-Star Reagent per cm2 of membrane. The Diluted CDP-Star Reagent is prepared by diluting the 25 mM CDP-Star stock solution with the 1X CDP-Star Dilution Buffer.

      Sample calculation
      100 cm2 x 0.025 ml/cm2 = 2.5 ml of diluted CDP-Star Reagent

      For maximum sensitivity
      Dilute 25 μl of the 25 mM CDP-Star Reagent to 2.5 ml with 1X CDP-Star Dilution Buffer.

      For maximum volume

      Dilute 5 μl of the 25 mM CDP-Star Reagent to 2.5 ml with 1X CDP-Star Dilution Buffer.

      C. Add the diluted CDP-Star Reagent to the hybridization bag.  Incubate for 5 minutes at room temperature with moderate shaking.

    8. Expose Membrane

      Open bag and drain as thoroughly as possible.  Smooth out any wrinkles or air bubbles and reseal the bag.  Clean the outside of the bag.  It is important to have close, uniform contact between the film and the membrain, and to make sure the DNA side of the membrane is towards the film, in order to obtain sharp images.  Expose the membrane to x-ray film for 15 seconds to 15 minutes, or a phospho-imager according to the manufacturer's recommendations.

      Note: Volumes required for detections done in a container, or a bag, that is considerably larger than the membrane may be greater.  Be sure that the membrane stays completley wetted.