NEB Phototope detection with CDP-Star

Introduction

Phototope detection with CDP-Star is based on chemiluminescence, an enzyme-catalyzed reaction which emits a blue light. This detection method is replacing radioactive detection due to sensitivity, convenience, safety and cost.
In the Phototope Detection Kits for nucleic acids, biotin associated with the target DNA provides the handle for the chemiluminescent detection. Biotinylated DNA is detected on a membrane support by first exposing the membrane to streptavidin, which binds to the biotinylated DNA. Next, biotinylated alkaline phosphatase is added, which binds to the streptavidin, resulting in the creation of a conjugate between the alkaline phosphatase and the DNA on the membrane. In the final step, the CDP-Star reagent is added. Alkaline phosphatase catalyzes the removal of the phosphate from the CDP-Star (phenylphosphate substituted 1,2 dioxetane) to yield a moderately stable intermediate which then spontaneously decays, emitting light at 461 nm. The emitted light is detected by exposing the membrane to x-ray film for 1 to 10 minutes.
The biotin handle can be associated with your target DNA either directly (covalently) or by hybridization. Biotin can be incorporated directly into DNA by using enzymatic polymerization of DNA with a biotinylated primer (for DNA sequencing) or by poly-merization in the presence of biotinylated nucleotide triphosphates (as with the NEBlot® Phototope System).
Protocol Overview
There are four basic steps for chemiluminescent detection using the Phototope-Star Detection Kit.

1. Target DNA is immobilized on a membrane support or hybridized to DNA crosslinked on a membrane support.

2. Streptavidin is bound to the biotinylated target DNA.

3. Biotinylated alkaline phosphatase is bound to streptavidin.

4. CDP-Star is reacted with the bound alkaline phosphatase and the emitted light is captured on x-ray film.

Kit Components

Streptavidin: 1.0 mg streptavidin at a concentration of 1.0 mg/ml (1000X).
Biotinylated Alkaline Phosphatase: 0.5 mg at a concentration of 0.5 mg/ml (1000X).
CDP-Star: 1.0 ml of a 25 mM solution.
CDP-Star Assay Buffer: 20 ml of 2-amino-2-methyl-1-propanol buffer supplied as a 25X liquid concentrate.

Additional Materials and Equipment for Detection

Solutions
Consult Appendix A for solution compositions and preparation procedures.
Blocking Solution
- 5% SDS, 125 mM NaCl
- 25 mM sodium phosphate
- pH 7.2
Wash Solution I
- 0.5% SDS, 12.5 mM NaCl
- 2.5 mM sodium phosphate
- pH 7.2
Wash Solution II
- 10 mM Tris-HCl
- 10 mM NaCl
- 1 mM MgCl2
- pH 9.5 (Stock can be made 10X)
Materials and Equipment
Membrane: Biotinylated target DNA blotted or hybridized onto appropriate membrane (see appendix B for membrane recommendations)pH meter
Heat sealer
Hybridization bags or containers
Platform shaker
x-ray film and exposure cassette

Phototope Detection Protocol

The following protocol will allow detection of biotinylated DNA on dot blots, Southern and Northern blots or sequencing membranes. Modifications for detection of plaque lifts and colony hybridizations are detailed in Appendix C. Examples of the detection volumes necessary for a 10 cm X 10 cm membrane sealed in a hybridization bag are given.
Note: In all steps during the detection procedures, it is important that there be sufficient room in the bag for the membrane to float free in solution. The hybridization bag must also be free of trapped air to ensure maximum contact between the reagents and the membrane.

1. Blocking stepAdd to the hybridization bag 0.1 ml of Blocking Solution per cm2 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 incubationDetermine the necessary volume of diluted solution based on using 0.05 ml of diluted streptavidin per cm2 of membrane. Prepare this volume by diluting the stock solution with Blocking Solution to a final concentration of 1 µg streptavidin/ml (1:1000 dilution). Incubate for 5 minutes at room temperature with moderate shaking. Drain the solution before next step.

Sample calculation: 100 cm2 X 0.05 ml/cm2 = 5 mlDilute 5 µl of streptavidin stock solution to 5 ml with blocking solution.

3. Wash 2 times p; Wash Solution IUse 0.5 ml Wash Solution I 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 incubationDetermine the necessary volume of diluted phosphatase based on using 0.05 ml of diluted phosphatase per cm2 of membrane. Prepare this volume by diluting the stock solution 1:1000 with Blocking Solution to a final concentration of 0.5 µg/ml. Incubate for 5 minutes at room temperature with moderate shaking. Drain the solution before next step.

Sample calculation: 100 cm2 X 0.05 ml/cm2 = 5 mlDilute 5 µl BAP stock solution to 5 ml with Blocking Solution

5. Wash 1 time in Blocking Solution Use 0.5 ml Blocking Solution 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 - Wash Solution IIUse 0.5 ml of 1X Wash Solution II 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 DNAThe CDP-Star reagent is supplied as a 25 mM stock solution and should only be diluted immediately before use. CDP-Star Diluent is supplied as a 25X stock solution.

CDP-Star stock solution can be diluted from 1:100 to 1:500* depending on its intended use. For experiments requiring maximum signal intensity and signal longevity, dilute 1:100 with 1X CDP-Star Diluent. For maximum volume in experiments where sensitivity is not a problem, dilute up to 1:500 with 1X CDP-Star Diluent. 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.

For Maximum Sensitivity

a. Dilute the 25X CDP-Star Assay Buffer with Milli-Q water to a 1X concentration. 1X CDP-Star Diluent 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 needed and prepare this quantity immediately before use. Use 0.025 ml of diluted CDP-Star reagent per cm2 membrane. The diluted CDP-Star reagent is prepared by diluting the 25 mM CDP-Star stock solution with the 1X CDP-Star Diluent.

Sample calculation:100 cm2 X 0.025 ml/cm2 = 2.5 ml of diluted CDP-Star reagent. Dilute 25 µl of 25 mM CDP-Star stock solution to 2.5 ml with 1X CDP-Star Diluent.

c. Add the diluted CDP-Star reagent to the hybridization bag. Incubate for 5 minutes at room temperature with moderate shaking. Open bag and drain as thoroughly as possible.

d. After draining the majority of the reagent off the membrane, smooth out any wrinkles and reseal the bag. Remove any liquid from the outside of the bag and expose to x-ray film. It is important to achieve close, uniform contact between the film and the membrane to obtain sharp images.

For Maximum Volume

Sample calculation:100 cm2 X 0.025 ml/cm2 = 2.5 ml of diluted CDP-Star reagent. Dilute 5 µl of 25 mM CDP-Star stock solution to 2.5 ml with 1X CDP-Star Diluent.

c. Add the diluted CDP-Star reagent to the hybridization bag. Incubate for 5 minutes at room temperature with moderate shaking. Open bag and drain as thoroughly as possible.

Troubleshooting

Low or Inconsistent Signal (Sensitivity)
Cause Remedy
Non-uniform distribution Ensure that membrane floats freely in
detection of detection reagents reagents and
that there are no bubbles.
Non-uniform contact Flatten bag and re-expose.between membraneand film
Incorrect CDP-Star pH Check pH of 1X CDP-Star Diluent and adjust to 9.5.
Underexposed film Re-expose for a longer period of time.

Uniform or Uneven High Background
Cause Remedy
Overexposed film Re-expose for shorter time period.
Inadequate washing Increase the time or the volume in the washing steps, particularly
after the addition of streptavidin.
Contaminated detection Remake solutions. Filter sterilize before storage.
solutions
Cause Remedy
Membrane drying out Rinse the membrane with Wash Solution II
during contact with film and reapply CDP-Star reagent. Expose in sealed bag.
Static electricity Static charge between the membrane and film can
result in dark lines.
Non-uniform distribution Ensure that membrane floats freely in detectionof detection reagents reagents and that there are no bubbles.
Impure water Use only Milli-Q or equivalent purity water for all
detection reagents.

Appendix A: Solution Compositions and Preparation

All solutions should be made with Milli-Q water (18 megohm-cm) or double deionized water.
Blocking Solution:

5% SDS, Phosphate, pH 7.2 (17 mM Na2HPO4, 8 mM NaH2PO4), 125 mM NaCl

Dissolve 7.3 g NaCl, 2.41 g Na2HPO4 (dibasic), 0.96 g NaH2PO4 (monobasic), and 49.89 g SDS in 800 ml water. Adjust the volume to 1 liter.

Wash Solution I:

0.5% SDS, Phosphate (1.7 mM Na2HPO4, 0.8 mM NaH2PO4), 12.5 mM NaCl

Dilute Blocking Solution 1 to 10 in water.
Wash Solution II (10X):

100 mM Tris-HCI, 100 mM NaCl, 10 mM MgCl2, pH 9.5

Dissolve 12.1 g Tris, 5.85 g NaCl, 2.03 g MgCl2 in 800 ml water. Adjust the pH to 9.5 with HCI. Adjust volume to 1 liter. Use at 1X.

Appendix B: Membrane Recommendations for the Phototope Detection Kit

Phototope Chemiluminescent Kits were developed using Immobilon-S membranes (Millipore, Inc.). For some applications, other membranes may also work equally well. Our recommendations for membranes follow. Please directly contact the supplier to order the membrane of your choice. Nitrocellulose membranes (any source) are not recommended for any Photope-Star Detection Kit application. New England Biolabs does not test, nor can take responsibility for, any particular membrane lot from any supplier. Membrane questions should be addressed to the supplier of the membrane.
Telephone/FAX (USA) numbers for membrane ordering/technical support:
CUNO: Telephone (800)-231-2259; FAX (203) 238-8716
Millipore: Telephone (800) 225-1380; FAX (617) 275-8200
MSI: Telephone (800) 444-8212; FAX (508) 366-5840
Pall (VWR Scientific): Telephone (800) 225-4290; FAX (617) 329-6522
For Southern blots, Northern blots, plaque lifts or colony hybridizations use neutral or non-charge modified nylon membranes.

(Supplier) MembraneCatalog Number Description(Millipore) Immobilon-SMBBU IMS02 10-pack, 15 cm X 20 cm MBBU IMSR0 roll, 12 in. X 50 ft MBBU IMS82 82 mm diameter discs (50) MBBU IMS32 132 mm diameter discs (50)(MSI) MagnaNO4HY320F5 5-pack, 20 cm X 20 cm NO4HY00010 roll, 30 cm X 3 m N04HY08250 82 mm diameter discs (50) N04HY13750 137 mm diameter discs (50)(Pall, VWR) Biodyne A28152-406 5-pack, 22 cm X 22 cm 28152-412 roll, 30 cm X 3 m 28152-400 82 mm diameter discs (25) 28152-403 137 mm diameter discs (25)(CUNO) Zetabind N5KNM508-01-045N5K 15-pack, 20 cm X 20 cm NM802-01-045N5 roll, 30 cm X 3 m NM908-01-045N5K 82 mm diameter discs (50) NM914-01-045N5K 132 mm diameter discs (50)Zetabind NUNM508-01-045NU 15 pack, 20 cm X 20 cm NM802-01-045NU roll, 30 cm X 3 m NM908-01-045NU 82 mm diameter discs (50) NM914-01-045NU 132 mm diameter discs (50)

Appendix C:Plaque Lifts and Colony Hybridizations

Screening libraries for clones containing specific nucleic acid sequences is often a fundamental step in cloning experiments. To identify the plaques and colonies which harbor the recombinant phage DNA or plasmids, the DNA is transferred to a membrane filter. The membrane with the lifted DNA is then hybridized with a biotinylated probe and positive plaques or colonies are identified by chemiluminescent detection.
This appendix contains modifications to the chemiluminescent detection protocol which enable detection of plaque lifts and colony hybridizations. Although the procedures are very similar to the standard protocol, some differences do exist. Therefore, it is important to read these instructions carefully before beginning.

Detection of Plaque Lifts and Colony Hybridizations

Lift the plaques or colonies onto neutral or non-charge modified nylon membranes specified in Appendix B. Hybridize the transferred DNA to a biotinylated probe by any standard method, then detect the DNA as follows:Detection can be performed with 5-6 membranes together provided that all washes and incubations are carried out in containers where each membrane floats freely. Detection in hybridization bags requires that membranes be placed back to back, two per bag. The volume of solution needed must be based on the total surface area of all the membranes in the bag.

1. Follow the standard protocol, with the following two exceptions: after incubation with streptavidin, wash four times, 5 minutes each, with 0.25 ml of Wash Solution I per cm2 of membrane; after incubation with biotinylated alkaline phosphatase, wash with blocking solution as described, then wash four times, 5 minutes each, with 0.25 ml of Wash Solution II per cm2 of membrane. The revised steps are outlined below with revisions in bold.

Detection Steps (volumes optimized for hybridization bags)

Repeats Reagent Time (min.) ml/cm2

1 Blocking reagent 5 0.1

1 Streptavidin 5 0.05

4 Wash Solution I 5 each 0.25

1 Biotin. Alk. Phos. 5 0.05

1 Blocking reagent 5 0.1

4 Wash Solution II 5 each 0.25

1 1X Substrate 5 0.025

2. After incubating with substrate, remove the membranes from the bag and place side by side in another bag. Place a piece of standard x-ray film on the membrane and expose for 5 minutes or an optimized time.

References

1. Feinberg, A.P. and Vogelstein, B. (1983) Anal. Biochem. 132, 6-13.
2. Feinberg, A.P. and Vogelstein, B. (1983) Anal. Biochem. 137, 266-267.
3. Denhardt, D.T. (1966) Biochem. Biophys. Res. Commun. 23, 641.
4. Bronstein, I., Olesen, C.E.M., Martin, C.S., Schreider, G., Edwards, B., Sparks, A. and Voyta, J.C. (1994) Bioluminescence and Chemiluminescence: Fundamentals and Applied Aspects (Campbel, A.H., Kricka, L.J. and Stanley, P.E., eds.) John Wiley and Sons, Chichester, England, 269-272.
5. Sambrook, J., E.F. Fritsch, T. Maniatis (1989) Molecular Cloning: A Laboratory Manual Second Edition 9.31-9.62 Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
6. Ausubel, F.M., R. Brent, R.E. Kingston, D.D. More, J.G. Seidman, J.A. Smith, and K. Struhl (1989) Current Protocols in Molecular Biology Wiley-Interscience, New York, New York.

The following are registered trademarks of New England Biolabs, Inc: CircumVent®, NEBlot®, Phototope®. CDP-Star is a trademark of Tropix, Inc. Magna is a trademark of MSI. Biodyne is a trademark of Pall. Zetabind is a trademark of CUNO.Immobilon is a trademark of Millipore Corp.LumiGLO® is a trademark of KPL.