The NEBlot Phototope Kit

Introduction


The NEBlot Phototope Kit incorporates biotin into hybridization probes through a random primer reaction. These biotinylated probes are hybridized to target nucleic acid immobilized on a membrane, and the target nucleic acid is detected through an enzyme catalyzed light-emitting reaction.

Protocol Overview

There are five basic steps in the Southern or Northern blotting procedures with the NEBlot Phototope Kit.

  • 1. Agarose Gel Electrophoresis of Nucleic AcidsSeparate the nucleic acid fragments by standard agarose gel electrophoresis.
  • 2. Transfer and ImmobilizationTransfer the nucleic acids to nylon membrane by standard Southern or Northern blotting. To covalently bind the nucleic acids, cross-link with UV light.
  • 3. Biotinylation of ProbesUse the NEBlot Phototope Kit to generate stable, highly-biotinylated probes. Biotinylated random octamers serve as the primers and biotinylated dATP is incorporated during the labeling reaction.
  • 4. HybridizationHybridize the biotinylated probe to the target DNA/RNA under standard conditions.
  • 5. Chemiluminescent DetectionUse the Phototope-Star Detection Kit for Nucleic Acids (NEB# 7020) to perform the chemiluminescent reactions. Streptavidin and biotinylated alkaline phosphatase are bound to the biotinylated probes through sequential incubations. The chemiluminescent substrate is added and the emitted light is captured on X-ray film.
    •

    Kit Components

    NEBlot Phototope Kit

    5X Labeling Mixture: (0.30 ml)Biotinylated random octamers in 5X labeling buffer.
    dNTP Mixture: (0.125 ml)1 mM dCTP, 1 mM dGTP, 1 mM TTP, 1 mM dATP/Biotin-14-dATP.
    Klenow Fragment (3´ - 5´ exo-): (0.025 ml, 5 units/µl)
    Unbiotinylated Control DNA: (0.02 ml, 0.5 mg/ml) Hind III digested Lambda DNA.
    Pre-biotinylated Molecular Weight Markers: (0.02 ml)A photobiotinylated mixture of Hind III digested Lambda DNA and Hae III digested fX174 DNA at a concentration of 100 ng/µl.
    Nuclease free dH2O: (1.0 ml)DEPC treated, autoclaved Milli-Q water.

    Additional Materials Required

    Solutions

    Consult Appendix A for solution compositions and preparation procedures.
    Southern Transfer and Plaque/Colony Lifts
    - 20X SSC Buffer (3 M NaCl, 0.3 M Na Citrate, pH 7.0)
    - Denaturation Solution (0.5 N NaOH, 1.5 M NaCl)
    - Neutralization Solution (1 M Tris-HCl, 1.5 M NaCl, pH 7.5)
    - 1X SSC, 1 M NH4OAc
    - Proteinase K Solution (100 µg/ml Proteinase K in 2X SSC)
    - PMSF Solution (1 mM PMSF in 2X SSC)
    Northern Transfer
    - 50 mM NaOH, 100 mM NaCl
    - 100 mM Tris-HCl, pH 7.5
    Probe Preparation
    - 0.2 M EDTA, pH 8.0
    - 4 M LiCl
    - Ethanol
    - 1X TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
    Hybridization and Washes
    - Prehybridization Solution (6X SSC, 5X Denhardt's reagent, 0.5% SDS, 100 µg/ml Denatured salmon sperm DNA)
    - 2X SSC, 0.1% SDS
    - 0.1X SSC, 0.1% SDS
    Formamide Hybridization
    - 20X SSPE Buffer (3M NaCl, 200 mM sodium phosphate, 20 mM EDTA, pH 7.4)
    - Formamide Prehybridization Solution (4X SSPE, 1% SDS, 5X Denhardt's reagent, 1 mg/ml total yeast RNA, 50% formamide)
    - Formamide Hybridization Solution (denatured probe in 5% dextran sulfate, 4X SSPE, 1% SDS, 5X Denhardt's reagent, 50% formamide)
    - 2X SSPE, 0.1% SDS
    - 0.1X SSPE, 0.1% SDS
    Stripping
    - 0.4 N NaOH, 0.1% SDS
    - 0.2 M Tris-HCl, 0.1X SSC
    - 2 mM EDTA, 0.1% SDS

    Materials and Equipment

    Membrane: Millipore Immobilon-S or other nylon blotting membrane with neutral or slight positive charge (see Appendix B for membrane recommendations)
    pH meter
    Oven
    Transilluminator or UV light
    Radiometer
    Plastic wrap
    Whatman 3MM paper
    Vacuum blotter or other blotting device
    Absorbent paper
    Heat sealer
    Hybridization bags
    Hybridization oven
    Platform shaker

    Blotting and Immobilization

    There are many protocols available for the transfer of nucleic acids to membranes. The following protocol has been optimized for use with the NEBlot Phototope Kit.
    Note: The NEBlot Phototope Kit was developed using an Immobilon-S membrane. Other high quality nylon membrane with neutral or slight positive charge may be substituted (see Appendix B for membrane recommendations).
    Note: Always wear powder-free gloves when handling the membrane and treat the membrane with care. Touching the membrane with bare hands will introduce oils and bacteria resulting in dark background spots. Mishandling may introduce background lines, spots, scrape marks, etc.

    Southern Blotting

    1. Perform agarose gel electrophoresis using standard techniques (4,5). Run 1-2 µl of pre-biotinylated markers on the gel. If the target DNA concentration is low (e.g. when probing for a single copy mammalian gene), dilute the markers to reduce their intensity.
    2. After electrophoresis, photograph the gel and place on a UV box for a few minutes to allow the UV light to nick the DNA (this can assist in transfer).
    Caution: Exposure to UV radiation poses a significant health hazard. Use extreme caution when exposing gels. Wear UV protective goggles and shield exposed skin.
    Next, the DNA should be partially depurinated by acid hydrolysis.
    a. Following photography, depurinate DNA by soaking the gel in 0.25 M HCl for 30 minutes.
    b. Wash the gel in water for 15 minutes to remove excess acid.
    3. Denature the DNA by soaking in Denaturation Solution (0.5 N NaOH, 1.5 M NaCl) for 30 minutes.
    4. Neutralize the gel by soaking in Neutralization Solution (1 M Tris-HCl, 1.5 M NaCl, pH 7.5) for 15 minutes.
    5. Repeat Step 4.
    6. Cut a piece of membrane the size of the agarose gel. Pre-wet the membrane in 2X SSC.
    7. Transfer the DNA to the membrane using any standard blotting method (4,5). Use 2X SSC as the transfer buffer.
    8. Transfer for 1-20 hours, following the chosen transfer protocol.
    9. After transfer, carefully mark the DNA side of the membrane, and place the membrane DNA side up on a clean piece of Whatman 3MM paper.

    Northern Blotting

    1. To separate RNA, use standard techniques to perform agarose/formaldehyde gel electrophoresis (5,6).
    2. After electrophoresis, photograph the gel, then soak the gel for 20-30 minutes in 50 mM NaOH, 10 mM NaCl.
    3. Soak the gel for 20-30 minutes in 10 mM Tris-HCl, pH 7.5.
    4. Soak the gel for 20-30 minutes in 2X SSC.
    5. Cut a piece of membrane the size of the agarose gel. Pre-wet the membrane in 2X SSC.
    6. Transfer the RNA to the membrane using any standard capillary blotting method (5,6). Use 2X SSC as the transfer buffer.
    7. Transfer for 6-20 hours.
    8. After transfer, carefully mark the RNA side of the membrane, and place the membrane RNA side up on a clean piece of Whatman 3MM paper.

    Drying the Membrane

    Note: The membrane should be completely dry prior to exposure to UV light. This is crucial in controlling how much cross-linking occurs.

    1. To prevent excessive curling, paper clip all four corners of the membrane to the Whatman paper.
    2. Place the membrane, clipped to the Whatman paper, in an oven or incubator at 42-80°C. Allow the membrane to dry completely. This requires approximately 10 to 15 minutes in an incubator with a fan and good air circulation, or 30 minutes in an oven with no circulation. The membrane can also be conveniently dried overnight at room temperature. The membrane is dry when the sides begin to curl (even though the corners are clipped down).

    UV Cross-linking DNA/RNA onto Membrane

    After the membrane is dried, the DNA/RNA is cross-linked to the membrane by UV irradiation. This immobilization step is important for the success of the detection procedure. If cross-linking is insufficient, the nucleic acid can be washed off the membrane. If the nucleic acid is over cross linked, it becomes unavailable for hybridization. This is especially true for the smaller fragments (less than 500 bp).
    For critical applications, it is recommended that the power source be calibrated, and the membrane be exposed for the optimal time period based on the determined power of the source.
    Caution: Exposure to UV radiation poses a significant health hazard. Use extreme caution when exposing membranes. Wear UV protective goggles and shield exposed skin.

    1. The UV source should have sufficient power at 254 nm to generate a minimum of 100 µWatts/cm2. Most transilluminators have power outputs in the 800-1000 µWatts/cm2 range. Hand-held lamps will have slightly lower outputs. If desired, determine the exact output of your source as described in Appendix C.
    Note that transilluminators are calibrated at the glass surface, and hand-held lamps are calibrated at the distance at which the membrane will be exposed.
    2. Determine the optimal exposure time for your light source. Optimal exposure time in seconds = (33,000 µWatts/cm2)(1 sec)/(power output in µWatts/cm2)
    Sample Calculation for TransilluminatorIf the transilluminator has a power output of 1000 µWatts/cm2, the optimal exposure time is 33 seconds.(33,000)(1)/(1000) = 33 seconds
    Sample Calculation for Hand-held LampIf the hand-held lamp has a power output of 100 µWatts/cm2 measured at 25 cm, the optimal exposure time at 25 cm is 5.5 minutes.(33,000)(1)/(100) = 330 seconds or 5.5 minutes
    3. Place the membrane (DNA/RNA side down) directly on the surface of the clean, dry transilluminator, or place the membrane (DNA/RNA side up) at the appropriate distance from the hand-held lamp. Expose for the calculated optimal time period. If you choose not to calibrate the light source, you may expose for 20-40 seconds. However, be aware that uncalibrated exposure may yield less than optimal results.

    NEBlot Phototope Probe Labeling

    Random Primer Biotinylation Reactions

    The NEBlot Phototope Kit follows the method of Feinberg and Volgelstein (1,2) to generate biotinylated probes. Random biotinylated octamers are used to prime DNA synthesis in vitro from denatured double-stranded template DNA. Biotinylated dATP is also incorporated during the extension reaction. The resulting probes are highly biotinylated, and therefore provide superior sensitivity in nucleic acid detection.

    Determine Required Quantity of Probe

    Before proceeding with the protocol, determine how much biotinylated probe you will need for your particular hybridization conditions and gel size. To produce the correct quantity, you can adjust the amount of DNA used in each reaction, vary the reaction time or perform multiple reactions.
    The factors to take into account are: a) the biotinylation reaction yield, b) the gel size, and c) the hybridization time.
    a) Following the standard reaction protocol given on page 15, the amount of labeled DNA synthesized in relation to template DNA and a given reaction time can be determined from Table 1.
    b) Use 0.1 ml of hybridization solution per cm2 of membrane.
    c) Synthesized probe concentrations between 10 ng/ml and 100 ng/ml can be used successfully. 20 ng/ml has been found to be an optimal concentration for typical overnight hybridizations. Higher concentrations can be used to shorten the hybridization time, or when probing for single copy genes or rare mRNA species.
    Sample CalculationFor a 20 x 20 cm gel with overnight hybridization. 400 cm2 x 0.1 ml/cm2 x 20 ng/ml = 800 ng
    To produce 800 ng of probe, perform a 25 ng biotinylation reaction overnight or a 1 µg reaction for 0.5 hours.

    Labeling Protocol

    The template DNA should be linear, and, to enhance the specificity of the probe, the insert should be separated from the vector. It is essential that the template DNA be fully denatured in Step 2. If it is not, the amount of biotinylated probe will be greatly reduced. You can use 5 ng to 1 µg of DNA in Step 1 without varying the rest of the protocol.
    Note: Allow components to thaw on ice. Klenow polymerase, however, should be kept at  -20°C except when in use.

    1. Dilute 5 ng-1 µg of template DNA (1-34 µl) in nuclease free H2O to a total volume of 34 µl. For the control reaction, add 2 µl (1 µg) of unbiotinylated lambda control DNA to 32 µl nuclease free H2O.
    2. Denature in boiling water for 5 minutes.
    3. Quickly place on ice for 5 minutes.
    4. Centrifuge briefly at 4°C.
    5. Add the following reagents to the template and control tubes in the order listed. 10 µl of 5X labeling mix (contains biotinylated random octamers) 5 µl of dNTP mix (contains dNTPs and biotin-dATP) 1 µl of Klenow Fragment (3´-5´ exo-)
    6. Incubate at 37°C for the calculated reaction time.
    7. Terminate the reaction by adding 5 µl of 0.2 M EDTA, pH 8.0.
    8. Precipitate the probe by adding 5 µl of 4M LiCl and 150 µl of ethanol, incubating at -70°C for 30 minutes, spinning, and washing with 70% ethanol.
    9. Resuspend in 20 µl of 1X TE.

    Note: A typical reaction using 100 ng of template in a 2 hour reaction will have resulted in the generation of approximately 595 ng of biotinylated DNA. Therefore, the concentration after resuspension in 20 µl will be about 30 ng/µl.
    For long term storage, keep the resuspended probe in a -20°C freezer.

    Determination of Probe Quality

    The quality of the biotinylated probe is an important factor affecting the success of the overall experiment. Both the amount of labeled probe and the amount of biotin incorporated in the probe must be adequate for successful hybridization and detection. The following assay will verify production of high quality probes.

    1. Make serial 10-fold dilutions of the biotinylated probe, the biotinylated lambda control, and the pre-biotinylated markers. Dilute out to 10-6 in 0.1N NaOH.
    2. Spot these three dilution sets on a membrane.
    In the previous example (step 9), if you use 1 µl for each spot, each set except for the diluted markers will contain spots with approx. 30 ng, 3 ng, 300 pg, 30 pg, 3 pg, 300 fg, and 30 fg of DNA.
    3. Dry the membrane and cross-link the DNA as described on pages 11 and 12.
    4. Follow procedures in the Phototope Detection Kit or the Phototope-Star Detection Kit for Nucleic Acids (NEB# 7020) to detect the three dilution sets.
    5. The pre-biotinylated marker should be visible down to the 103-fold dilution. The biotinylated probe and the biotinylated lambda control should be visible within the 100 fg to 10 fg range to be considered sufficiently biotinylated.

    Determination of Probe Sensitivity

    1. Denature the target DNA by suspension in 0.1 N NaOH. Make several dilutions of the DNA down to 0.1 pg/ml.
    2. Spot 1 µl of each dilution directly onto membrane and allow to air dry for 15 minutes or until spots disappear.
    3. UV cross-link and hybridize with probe, follow standard hybridization protocols (see next section).
    4. Detect using protocols from the Phototope® Detection Kit or the Phototope®-Star Detection Kit for Nucleic Acids.

    Hybridization

    There are many methods to hybridize probes in solution to nucleic acids immobilized on membranes. Most of these standard methods can be adapted to work well for chemiluminescent detection.
    Note: Methods which include a blocking step with some form of dried milk should be avoided as the detection system may be sensitive to biotin naturally present in milk.
    Standard Hybridization
    The following method has been optimized for chemiluminescent detection on Immobilon-S membranes. These conditions may be altered in temperature, salt concentration, and/or time to suit the specific hybridization requirements of your particular probe and target sequences.

    1. Thoroughly wet both sides of the membrane in 6X SSC.
    2. Place the membrane in a hybridization bag and prehybridize for 1 hour at 68oC (55°C Blaxter lab) in Prehybridization Solution. Use approximately 0.1 ml of solution per cm2.
    3. Denature the biotinylated probe by placing in boiling water for 5 minutes, chilling on ice for 5 minutes, and centrifuging briefly at 14,000 X g.
    4. Hybridize using the same solution and volume used in Step 2, but include approximately 20 ng/ml of denatured probe (probe DNA can be added to the prehybridization solution already in the bag). Allow hybridization to continue for 6 hours to overnight at 68oC (55°C Blaxter lab). If a greater concentration of probe is used, the hybridization can be shortened.
    5. After hybridization, remove the membrane from the bag and wash in 2X SSC, 0.1% SDS at room temperature for 5 minutes. Repeat.
    6. Wash the membrane in 0.1X SSC, 0.1% SDS at 68°C (55°C Blaxter lab) for 15 minutes or vary temperature for desired stringency. Repeat.
    7. Place the washed membrane in a new hybridization bag for subsequent chemiluminescent detection.

    Formamide Hybridization

    1. Thoroughly wet both sides of the membrane in 4X SSPE.
    2. Place the membrane in a hybridization bag and prehybridize for 2 hours at 50°C in Formamide Prehybridization Solution. Use approximately 0.1 ml of solution per cm2.
    3. Denature the probe by placing in boiling water for 5 minutes, chilling on ice for 5 minutes and centrifuging briefly at 14,000 X g.
    4. Add 20 ng/ml denatured probe to Formamide Hybridization Solution and hybridize at 50°C for 6-20 hours.
    5. After hybridization, remove the membrane from the bag and wash in 2X SSPE, 0.1% SDS at room temperature for 5 minutes. Repeat.
    6. Wash the membrane in 0.1X SSPE, 0.1% SDS at 68°C for 5 minutes. Repeat.
    7. Rinse the membrane briefly in 2X SSPE, 0.1% SDS and place in a new hybridization bag for subsequent chemiluminescent detection.

    Storing the membrane

    The membrane can be stored in a small volume of 1X SSC or 1X SSPE in the sealed bag at room temperature for extended periods. Do not allow the membrane to dry out.

    Stripping and Reprobing

    After detection with one probe is complete, the membrane can be stripped and washed to remove the probe. Hybridization with another probe can then be performed beginning with the prehybridization step.
    For Southerns

    1. Rinse the membrane in water.
    2. Incubate in 0.4 N NaOH, 0.1% SDS at 25°C (70°C Blaxter lab) for 30 minutes.
    3. Rinse membrane in 0.2 M Tris-HCI, 0.1X SSC for 30 minutes at 25°C.
    4. The membrane can now be reprobed.

    For Northerns

    1. Rinse the membrane in water.
    2. Incubate in a large volume (~ 500 ml) of 2 mM EDTA, 0.1% SDS at 80°C for 15 minutes.
    3. Rinse membrane in 2X SSC at room temperature.
    4. The membrane can now be reprobed.

    Note: Washing for more than half an hour (overstripping) may adversely affect the binding of RNA or DNA to the membrane.

    Troubleshooting

    Low or Inconsistent Signal after Chemiluminescent Detection

    Cause Remedy
    Problems with Probe
    Insufficient biotin-labeling Check the level of probe biotinylation by comparing
    to biotinylated control DNA and prebiotinylated
    markers. If the probe labeling looks weak but the
    biotinylated control looks fine, the template DNA is
    the problem. Be sure template is adequately purified.
    Confirm that the template is linear and completely heat
    denatured before biotinylation. If both the probe and
      biotinylated control look weak compared to pre-
    biotinylated markers, biotinylate again with fresh
    reagents. Check detection reagents.
    Loss of probe during Add some carrier DNA or RNA during precipitation.
    precipitation

    Problems with Blotting
    Transfer inadequate Review blotting steps. Make sure there is good
    contact (no bubbles) between membrane and gel.
    Include a lane of pre-biotinylated markers in the gel to aid in
    troubleshooting.
    Cross-linking inadequate Ensure that the membrane is completely dry
    before cross-linking. Calibrate light source and expose the
    membrane for the optimal time period as calculated on page 12.

    Problems with Hybridization and Detection
    Cause Remedy
    Inefficient hybridization Increase probe concentration during hybridization.
    Adjust the temperature, salt concentrations, or
    hybridization times to reflect the percent homology
    between the probe DNA and the target nucleic acid.
    Spot a dilution of the probe DNA on the membrane
    as an aid for troubleshooting.
    Nonuniform distribution Ensure that membrane floats freely in detection
    of detection reagents reagents and that there are no bubbles.
    Nonuniform contact Flatten bag and re-expose.
    between membrane and film
    Underexposed film Re-expose for a longer period of time.

    Uniform or Uneven High Background
    Cause Remedy
    Problems with Probe
    Impure template DNA Repurify if necessary to reduce nonspecific
    hybridization.
    Problems with Blotting
    Fingerprints, scraped or Always wear powder-free gloves and handle
    damaged membranes membranes carefully.
    Contaminated transfer Remake solutions. Filter sterilize before storage.
    solutions

    Problems with Hybridization and Detection
    Excess probe Reduce concentration of probe in hybridization.
    Overexposed film Reexpose 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 Refer to appropriate Phototope detection manual.
    Membrane drying out Rinse the membrane with Wash Solution II and during contact with
    film reapply chemiluminescent reagent. Expose in
    sealed bag.
    Static electricity Static charge between the membrane and
    film can result in dark lines.
    Nonuniform distribution Ensure that membrane floats freely in detection
    of detection reagents reagents and that there are no bubbles.

    Appendix A:Solution Compositions and Preparation

    Make solutions with Milli-Q water (18 megohm-cm) or ultra pure double deionized water.

    20X SSC: 3 M NaCl, 0.3 M NaCitrate, pH 7.0

    Dissolve 175.3 g NaCl and 88.2 g sodium citrate in 800 ml water. Adjust the pH to 7.0 with a few drops of 10 N NaOH. Adjust the volume to 1 liter. Sterilize by autoclaving.
    For various dilutions of SSC, dilute 20X SSC with the appropriate amount of water.

    Denaturation Solution: 0.5 N NaOH, 1.5 M NaCl

    Dissolve 20 g NaOH and 87.7 g NaCl in 800 ml water. Adjust final volume to 1 liter.

    Neutralization Solution: 1 M Tris-HCl, 1.5 M NaCl, pH 7.5

    Dissolve 121.1 g Tris and 87.7 g NaCl in 800 ml water. Adjust pH to 7.5 with HCI. Adjust volume to 1 liter.

    1X SSC, 1 M NH4OAc

    Dissolve 77.1 g Ammonium acetate in 800 ml water. Add 50 ml 20X SSC and adjust final volume to 1 liter.

    Proteinase K Stock Solution (20 mg/ml)

    Dissolve 200 mg Proteinase K in 10 ml water. Store frozen as aliquots. Thaw and dilute as needed.

    Proteinase K Solution (100 µg/ml Proteinase K in 2X SSC)

    Mix 1 ml of 20 mg/ml Proteinase K Stock Solution, 20 ml of 20X SSC and 179 ml water.

    *PMSF Stock Solution (200 mM in propanol)

    Dissolve 0.35 g solid PMSF in 10 ml 2-propanol. Heat to 50°C to dissolve. Store at -20°C. Redissolve by heating for dilution as needed.

    *PMSF Solution (1 mM PMSF in 2X SSC)

    Prepare fresh, just prior to use. Mix 1 ml of PMSF Stock Solution, 20 ml of 20X SSC and 179 ml of water.

    50 mM NaOH, 0.1 M NaCl

    Add 5 ml 10 N NaOH to 800 ml water. Add 5.85 g NaCl. Adjust volume to 1 liter.

    1.0 M Tris-HCl Stock Solution:

    Dissolve 121.1 g Tris in 800 ml water. Adjust the pH to 7.5 with HCI. Adjust volume to 1 liter.

    0.5 M EDTA Stock Solution:

    Add 186.1 g Na2EDTA·2H2O to 800 ml water. Stir vigorously. Adjust the pH to 8.0 with NaOH (about 20 g). Adjust volume to 1 liter.

    4 M LiCl

    Dissolve 17 g LiCl in 80 ml water. Adjust volume to 100 mls.

    TE:

    10 mM Tris, 1 mM EDTA, pH 8.0
    Mix 10 ml of 1 M Tris-HCl stock solution (pH 8.0), 2 ml of 0.5 M EDTA stock solution, and 988 ml of water.

    Prehybridization Solution: 6X SSC, 5X Denhardt's Reagent, 0.5% SDS, 100 µg/ml Denatured Salmon Sperm DNA

    Mix 300 ml 20X SSC, 100 ml 50X Denhardt's, and 50 ml 10% SDS. Dilute to 1 liter with water. Immediately prior to use, denature the 10 mg/ml salmon sperm DNA stock solution by heating for 5 minutes in boiling water. Add the appropriate amount of denatured salmon sperm DNA to result in a final concentration of 100 µg/ml.

    50X Denhardt's Reagent:

    Dissolve 5 g ficoll, 5 g polyvinylpyrrolidone, and 5 g bovine serum albumin in water. Dilute to 500 ml.

    10% Sodium Dodecyl Sulfate (SDS):

    Dissolve 100 g electrophoresis grade SDS in 900 ml water. Heat to 68°C to assist dissolution. Adjust the pH to 7.2 by adding a few drops of concentrated HCl. Adjust the volume to 1 liter. (Caution: Wear a mask when weighing SDS and wipe down the weighing area and balance after use).

    10 mg/ml Salmon Sperm DNA:

    Dissolve 500 mg of salmon sperm DNA or other nonspecific DNA in 40 ml of water. Stir for several hours to solubilize. Adjust volume to 50 ml. Sonicate or shear the DNA to generate fragments. Aliquot and store frozen.

    2X SSC, 0.1 % SDS

    Mix 100 ml 20X SSC, 10 ml 10% SDS, and 890 ml water.

    0.1X SSC, 0.1% SDS

    Mix 5 ml 20X SSC, 10 ml 10% SDS, and 985 ml water.

    20X SSPE:

    3 M NaCl, 200 mM Sodium Phosphate, 20 mM EDTA, pH 7.4
    Dissolve 175.3 g NaCl and 27.6 g NaH2PO4 in 700 ml water. Adjust pH to 7.4 with 10 N NaOH. Add 40 ml of 0.5 M EDTA and adjust volume to 1 liter with water. Adjust pH to 7.4. Sterilize by autoclaving.

    Formamide Prehybridization Solution:

    4X SSPE, 5X Denhardt's Reagent, 1.0% SDS, 1 mg/ml Total Yeast RNA, 50% Formamide
    Mix 200 ml 20X SSPE, 100 ml 50X Denhardt's and 50 ml 10% SDS. Dilute to 500 ml with water. Add 500 ml Formamide and mix. Immediately before use, dissolve total yeast RNA in desired volume to 1 mg/ml.

    Formamide Hybridization Solution:

    5% Dextran Sulfate, 4X SSPE, 5X Denhardt's Reagent, 1.0% SDS, 50% Formamide
    Mix 200 ml 20X SSPE, 100 ml 50X Denhardt's and 50 ml 10% SDS. Dilute to 450 ml with water and dissolve 50 g Dextran Sulfate. Adjust volume to 500 ml with water. Add 500 ml Formamide and mix. Immediately before use, calculate the necessary volume and add denatured probe to a final concentration of 20 ng/ml.

    2X SSPE, 0.1% SDS

    Mix 100 ml 20X SSPE, 10 ml 10% SDS and 890 ml water.

    0.1X SSPE, 0.1% SDS

    Mix 5 ml 20X SSPE, 10 ml 10% SDS and 985 ml water.

    0.4 N NaOH, 0.1% SDS

    Mix 40 ml 10 N NaOH stock solution, 10 ml 10% SDS, and 950 ml water.

    0.2 M Tris, 0.1X SSC

    Mix 200 ml 1 M Tris-HCl (pH 7.5) stock solution, 5 ml 20X SSC, and 795 ml water.

    2 mM EDTA, 0.1% SDS

    Mix 4 ml 0.5 M EDTA, 10 ml 10% SDS, and 986 ml water.

    Appendix B:Membrane Recommendations for the NEBlot Phototope 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 Phototope application. New England Biolabs does not test or take responsibility for any particular membrane 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)
    Membrane
    Catalog Number Description

    (Millipore)
    Immobilon-S
    MBBU 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)
    Magna
    NO4HY320F5 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 A
    28152-406 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 N5K
    NM508-01-045N5K 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 NU
    NM508-01-045NU 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: UV Light Source Calibration

    Calibration is important because different sources have different power outputs. The power output from a single source can vary with the age of the bulbs, and the type of filtering some sources use to reduce certain wavelengths.

    1. Allow the bulbs to warm up sufficiently to generate consistent power. Make sure the hand-held lamp is on the short wavelength setting.
    2. For transilluminators, place a radiometer on the glass surface. Measure the power output at 254 nm.
    3. For hand-held lamps, place the radiometer at a distance that will allow the entire surface of the membrane to be exposed when the lamp shines on it. This is very important. Since the power of the source decreases with the square of the distance to the membrane.
    4. If a radiometer is not available, estimate the power output of the source (consult the manual or contact the manufacturer) and expose a membrane for several different times to find the optimum cross-linking exposure time.

    Appendix D: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 procedures for performing plaque lifts and colony hybridizations using the NEBlot Phototope kit. Although the procedures are very similar to the Southern blotting methods, some differences do exist. Therefore, it is important to read these instructions carefully before beginning. Frequent reference is made to the Southern blotting section for previously established procedures.

    Transferring plaques to membranes

    1. Plate and incubate the transfected library according to standard procedures.
    2. Refrigerate the incubated plates for at least 30 minutes to harden the media.
    3. Label a dry membrane disk using a pencil (excess ink from ball point pens and felt tip pens can lead to high background).
    4. Using blunt-ended forceps, or wearing gloves, carefully place the membrane disk onto a plate.
    5. Once the membrane has become wet, let the plaques adsorb for 1 minute. Mark the membrane with syringe pricks to facilitate alignment after hybridization.
    6. Carefully remove the membrane from the plate using forceps.
    7. Up to 8 membranes can be taken from each plate, but increase the time on the plate by one minute per membrane (i.e. for the 8th membrane, leave on the plate for 8 minutes).
    8. Immerse the membranes in the following solutions:
    a. 1.5 M NaCl, 0.5 N NaOH for 2 minutes.
    b. Deionized water to remove excess NaOH (dip briefly).
    c. 1.5 M NaCl, 0.5 M Tris-HCl, pH 8.0 for 1 minute. Check the pH often, carry over of NaOH can rapidly shift the pH.
    d. 1.5 M NaCl, 0.5 M Tris-HCl, pH 8.0 for 1 minute. A second container of this solution ensures proper washing of the filters.
    e. 20X SSC for 1 minute.
    f. 1 M Ammonium acetate, 1X SSC for 1 minute. Rub the membrane with a gloved hand while it is in the last solution to remove cellular debris and agar.
    9. To remove excess liquid, place the membranes on Whatman® 3MM filter paper, DNA side up, and air dry for a few minutes.

    Drying and UV Cross-linking

    1. To prevent curling, paper clip the membranes at the four corners to a piece of Whatman 3MM filter paper. Dry in a 68°C oven for 10-15 minutes to remove all traces of ammonium acetate.
    2. UV cross-link according to procedures outlined previously.
    Proteinase K Treatment
    1. Immerse the membranes in Proteinase K solution and heat with gentle rocking for 1 hour at 55°C.
    2. Rinse briefly in 2X SSC.
    3. Immerse the membranes in PMSF Solution at room temperature with gentle rocking for 15 minutes.
    4. Rinse twice in 2X SSC.

    Biotin Labeling Probe

    Determine the amount of probe needed for your set of screening procedures. Follow the guidelines on page 14, with one important exception. For colony and plaque lifts, you will only need 0.02 ml of hybridization solution per cm2 of membrane (for Southerns and Northerns, the required volume is 0.1 ml/cm2).
    Sample Calculation:For five, 82 mm disks with overnight hybridization.
    Membrane area = 5 x ( x 4.12) = 264 cm2
    264 cm2 x 0.02 ml/cm2 x 20 ng/ml = 105.6 ng
    Biotin label the required amount of probe.
    Note: Multiple rounds of gel fragment purification will reduce vector background.

    Hybridization

    Note that 5-6 membranes can be hybridized per bag; 0.02 ml/cm2.

    Detecting the DNA

    Detection should be performed according to protocols provided with one of the Phototope Detection kits.

    Transferring Colonies to Membranes

    1. Spread the transformed bacteria on thick, well dried plates and incubate overnight at 37°C. Note that small colonies will yield better overall results than large colonies.
    2. Label a membrane disk using a pencil (excess ink from ball point pens and felt tip pens can lead to high background).
    3. Using blunt-ended forceps, or wearing gloves, wet the membrane by placing it on a blank plate.
    4. Place the wetted membrane onto a colony-containing plate and let colonies absorb for 1-2 min. Mark the membrane with syringe pricks to facilitate alignment after hybridization. The length of time you let the membrane stay on the plate depends on colony size, wetness of plate, and other indeterminate factors. It is often good to have a practice plate. If after 2 minutes, the colonies have not adhered to the membrane, use a round replica plating block to gently press on the membrane. If after 2 minutes, all the colonies have adhered to the membrane, leaving nothing on the plate, shorten the time the membrane is in contact with the plate.
    Note: Very little DNA is required on the membrane in order to see a signal.
    5. Carefully remove the membrane from the plate using forceps.

    Processing the Membranes

    Perform the following steps in a petri dish:

    1. Layer several sheets of Whatman 3MM filter paper on the bottom of the first petri dish and saturate with 1.5 M NaCl, 0.5 N NaOH.
    2. Place the membranes, colony side up, on the saturated filter paper for 5 minutes. Colonies will become glassy in appearance.
    3. Immerse the membranes in the following solutions:
    a. 1.5 M NaCl, 0.5 M Tris-HCl, pH 8.0 for 2-3 minutes. Check the pH often. Carry over of NaOH can rapidly shift the pH.
    b. 1.5 M NaCl, 0.5 M Tris-HCl, pH 8.0 for 2-3 minutes. A second container of this solution ensures proper washing of the filters.
    c. Place the membranes in 2X SSC, 0.1 % SDS. Remove bacterial debris by rubbing each membrane with gloved hands. This step is critical.
    d. Immerse the membranes in 1 M NH4OAc, 1X SSC for 1 minute.
    6. To remove excess liquid, place the membranes on a Whatman 3MM filter paper, DNA side up, for a few minutes.

    Drying and UV Cross-linking, Biotin Labeling Probe, Hybridizing, Detecting the DNA

    Follow the procedures as described below.

    Proteinase K Treatment

    1. Immerse the membranes in Proteinase K solution and heat with gentle rocking for 1 hour at 55°C.
    2. Rinse briefly in 2X SSC.
    3. Immerse the membranes in PMSF solution at room temperature with gentle rocking for 15 minutes.
    4. Rinse twice in 2X SSC.

    Appendix E: Probing with Biotinylated Oligonucleotides

    The NEBlot Phototope Kit will not efficiently label short (<100 bases) oligonucleotides. However, biotinylated oligonucleotides obtained from synthesis or end labeling can be used in conjunction with one of the Phototope Detection Kits.
    As an example, the pUC19 reverse sequencing primer (NEB #1233BT) which is supplied as 0.05 OD A260 units lyophilized, can be used. We recommend using 0.05 ml of hybridization solution per cm2 of membrane and 1.0 pmol of biotinylated probe per ml (this is excess for most purposes). Thus, for a 20 cm X 20 cm blot (400 cm2), one would use 20 ml of hybridization solution with 20 pmol of biotinylated probe.
    For this example, the lyophilized pUC19 primer (0.05 OD A260.i.);) should be dissolved in 0.5 ml of water, bringing the concentration to 3.5 pmol per µl. Hybridization should be carried out with 5.7 µl of probe. This probe concentration was optimized for probing of sequencing membrane, where literally thousands of bands are probed. Under standard Southern blot conditions, far less probe would be required.

    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. 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.
    5. 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 Phototope® Family of Chemiluminescent Kits

    NEBlot® Phototope® Kit #7550Random Primer Biotin Labeling of DNA for Northern and Southern Blotting
    CircumVent® Phototope® Kit #7430For Chemiluminescent Thermal Cycle DNA Sequencing
    Phototope®-Star Detection Kit for Nucleic Acids #7020(contain sufficient reagents for detection with CDP-Star on up to 20,000 cm2 of membrane)
    Phototope®-Star Detection Kits for Western Blotting(contain sufficient reagents for detection of Western blots with CDP-Star on up to 5,000 cm2 of membrane)
    anti-Rabbit IgG #7051
    anti-Mouse IgG #7052
    anti-Human IgG #7053
    Phototope®-HRP Detection Kits for Western Blotting(contain sufficient reagents for detection of Western blots with LumiGLO® on 5,000 cm2 of membrane)
    anti-Rabbit IgG #7071
    anti-Mouse IgG #7072
    anti-Human IgG #7073