EZ-Hybridization Solution

Cat. No.: 01-889-1B (100ml)

Storage: Room temperature For long term storage, store at 2-8ºC

 

Product Description

EZ-Hybridization Solution enables shorter hybridization times and decreases backgrounds. Therefore, low-copy RNA species on Northern blots and single-copy genes on Southern blots can be detected within 1-2 hours of hybridization using radioactively or non-radioactively labeled probes (compared to 12-24 hours with standard buffers).

cDNA Probes
1. General considerations

  • EZ-Hybridization Solution can be used for Southern blot, Northern blot, or colony hybridizations. The only difference between the protocols is the incubation temperature. Note: Use 68ºC for Northern; use 60ºC for Southern
  • The hybridization temperature used in the following protocols is suitable for hybridization of DNA probes of average G/C content (40%). The optimal temperature for probes of different G/C content must be determined empirically (Sambrook et al ., 1989)
  • The recommended final DNA probe concentration is 2-10ng/ml or 1-2x106 cpm/ml for Northern or Southern hybridizations. (Probe concentrations >10ng/ml will reduce the time needed for hybridization, but may increase background). For high target applications, e.g. colony hybridizations, lower probe concentration can be used.
  • Concentrated probe should not be added directly to the membrane because uneven concentrations may result in localized background.
  • EZ-Hybridization Solution allows shorter hybridization times, e.g. 1-2 hours. Nevertheless, overnight hybridization can still be performed.
    Note: Long hybridization time, combined with high probe concentration, may lead to high background.

2. Hybridization with EZ-Hybridization Solution with radioactively labeled probes

  • Warm the EZ-hybridization Solution at 68ºC (60ºC), and stir well to completely dissolve any precipitate.
  • Pre-hybridize membranes in a minimum of 0. 1ml/cm2 of EZ-Hybridization Solution with shaking at 68ºC (60ºC) for 30 minutes. The volume of solution must be sufficient to completely cover the membrane, or high backgrounds may result.
  • Denature the radioactively labeled DNA probe at 95-100ºC for 2-5 minutes. Chill quickly on ice.
  • Add radiolabeled probe to a sufficient volume of fresh EZ-Hybridization Solution. Mix gently. For recommended final concentration, see above (1.3).
  • Replace the EZ-Hybridization Solution with the fresh solution containing the radiolabeled DNA probe. Remove all air bubbles from the container, and make sure the EZ-Hybridization Solution is evenly distributed over the entire blot.
  • Hybridize with continuous shaking at 68ºC (60ºC) for 1-2.5 hours. (For high target applications, shorter hybridization times can be used. For single-gene sequences, hybridization can be performed overnight).
  • Rinse the blot in a large amount of 2xSSC, 0.1% SDS 2-3 times at room temperature. Was with at least 0.5ml/cm2 of 2xSSC, 0.1% SDS for 30-40 minutes with continuous agitation; replace the wash solution twice.
  • Wash the blot in at least 0.5ml/cm2 of 0.1xSSC, 0.1% SDS with continuous shaking at 65ºC (65º) for 40 minutes with two changes of fresh solution. (For certain RNA probes, washing temperature above 65ºC (up to 70ºC) may be necessary in order to obtain the correct stringency).
  • Remove the blot with forceps and shake off excess wash solution. Note: Do not blot-dry or allow the membrane to even partially dry. If the membrane is allowed to dry, subsequent removal of the probe from the membrane for re-probing may be difficult.
  • For 32p-labeled probes, immediately cover the blot with plastic wrap. Mount on Whatman paper (3MM). Wrap again with plastic wrap. For 35 s-labeled probes, autoradiograph dried membranes at room temperature without plastic wrap.
  • Expose to x-ray film at –70ºC with two intensifying screens.
  • If the membrane is to be re-probed, incubate the blot in sterile H2O containing 0.5% SDS as outlined below.
    Heat the sterile H2O/0.5% SDS solution to 90-100ºC.
    Remove plastic wrap from blot and immediately place in the heated solution. Make sure that exposure to air is minimal.
    Incubate for 10 minutes, shaking frequently.
    Allow the solution to cool for 10 minutes before removing the blot.
    Remove the blot and air-dry until it is dry enough to be slipped into plastic bag. The membrane can be stored at –20ºC until needed.

3. Hybridization using EZ-Hybridization Solution with non-radioactively labeled probes

  • Warm the EZ-Hybridization Solution at 68ºC (60ºC) and stir will to completely dissolve any precipitate.
  • Pre-hybridize membranes in a minimum of 0.1ml/cm2 of EZ-Hybridization Solution with continuous shaking at 68ºC (60ºC) for 30 minutes. The volume of solution must be sufficient to completely cover the membrane, or high backgrounds may result.
  • Denature the radioactively labeled DNA probe at 95-100ºC for 2-5 minutes. Chill quickly on ice.
  • Add non-radiolebeled probe to a sufficient volume of fresh EZ-Hybridization Solution. Mix gently. For recommended final probe concentrations, see above (1.3).
  • Replace the EZ-Hybridization Solution with the fresh solution containing the non-radiolabeled DNA probe. Remove all air bubbles from the container, and make sure the EZ-Hybridization Solution is evenly distributed over the entire blot.
  • Hybridize with continuous shaking at 68ºC (60ºC) for 1-2.5 hours. (For high target applications, shorter hybridization times can be used. For single-gene sequences, hybridization can be performed overnight).
  • Wash the membranes at room temperature twice, 15 minutes each time, with at least 0.5ml/cm2 of 2xSCC, 0.1% SDS.
  • Wash the membrane at 68ºC (60ºC) twice, 15 minutes each time, with at least 0.5ml/cm2 of 1-0.1xSSC, 0.1% SDS, with the continuous agitation.
    Note: These washing conditions may be too stringent for probes which are not completely homologous to the target. If this is the case, lower the temperature to 50ºC.
  • Remove the blot with forceps and shake off excess wash solution. Blots can then be used directly for chemiluminescent detection (EZ-ECL, Cat. No. 20-500-120) of hybridized DNA, or stored air-dried for later detection using colorimetric detection systems.

Oligonucleotide Probes
1. General considerations

  • EZ-Hybridization Solution can be used with oligonucleotide probes in Northern blot or Southern blot hybridization.
  • The hybridization temperature used in the following protocols is suitable for hybridization of oligonucleotide probes of average G/C content (40%). The optimal temperature for probes of different G/C content must be determined empirically (1).
  • The recommended final oligonucleotide probe concetration is 10-50ng/ml or 0.5-2x107 cpm/ml. Probe concentration of >50ng/ml will reduce the time needed for hybridization, but may increase background.
  • Concentrated probe should not be added directly to the membrane, because uneven concentrations may result in localized background.
  • EZ-Hybridization Solution allows shorter hybridization times, e.g. 30-60 minutes.

2. Hybridization with EZ-Hybridization Solution with radioactively labeled oligonucleotides

  • Warm the EZ-Hybridization Solution at 42ºC.
  • Pre-hybridize membranes in a minimum of 0.1ml/cm2 of EZ-Hybridization Solution with continuous shaking at 42ºC for 30 minutes. The volume of solution must be sufficient to completely cover the membrane, or high background may result.
  • Add radiolabeled probe to a sufficient volume of fresh EZ-Hybridization Solution. Mix gently. For recommended final probe concentrations, see above (1.3).
  • Replace the EZ-Hybridization Solution with the fresh solution containing the radiolabeled oligonucleotide probe. Remove all air bubbles from the container and make sure the EZ-Hybridization Solution is evenly distributed over the entire blot.
  • Hybridize with continuous shaking at 42ºC for 30-60 minutes. For high target applications, shorter hybridization times can be used. For single-gene sequences, hybridization can be performed overnight, but it can also lead to increased background.
  • Wash the membranes at room temperature, 20 minutes in 0.5ml/cm2 of 5xSCC, 0.1% SDS.
  • Wash the membranes at 42ºC, 15 minutes each time in 0.5ml/cm2 of 1-0.1xSSC, 0.1% SDS.
  • Remove the blot with forceps and shake off excess wash solution. Note: Do not blot-dry of allow the membrane to even partially dry. If the membrane is allowed to dry, subsequent removal of the probe from the membrane for re-probing may be difficult.
  • For 32p-labeled probes, immediately cover the blot with plastic wrap. Mount on Whatman paper (3MM). Wrap again with plastic wrap. For 35s-labeled probes, autoradiograph dried membranes at room temperature without plastic wrap.
  • Expose to x-ray film at –70ºC with two intensifying screens.
  • If the membrane is to be re-probed, incubate the blot in steril H2O containing 0.5% SDS as outlined below.
    Heat the sterile H2O/0.5% SDS solution to 90-100ºC.
    Remove plastic wrap from blot and immediately place in the heated solution. Make sure that exposure to air is minimal.
    Incubate for 10 minutes, shaking frequently.
    Allow the solution to cool for 10 minutes before removing the blot.
    Remove the blot and air-dry until it is dry enough to be slipped into a plastic bag. The membrane can be stored at –20ºC until needed.

3. Hybridization with EZ-Hybridization Solution with non-radioactively labeled oligonucleotides

  • Warm the EZ-Hybridization Soltution at 42ºC.
  • Pre-hybridize membranes in a minimum of 0.1ml/cm2 of EZ-Hybridization Solution with continuous shaking at 42ºC for 30 minutes. The volume of solution must be sufficient to completely cover the membrane, or high backgrounds may result.
  • Add non-radiolabeled probe to a sufficient volume of fresh EZ-Hybridization Solution. Mix gently. For recommended final probe concentration, see above (1.3).
  • Replace the EZ-Hybridization Solution with the fresh solution containing the non-radiolabeled oligonucleotide probe. Remove the air bubbles from the container and make sure the EZ-Hybridization Solution is evenly distributed over the entire blot.
  • Hybridize with continuous shaking at 42ºC for 30-60 minutes.
  • Wash the membranes at room temperature for 30 minutes with a minimum of 0.5ml/cm2 of 5xSSC, 0.1% SDS, with continuous agitation. Replace the wash solution once.
  • Wash the membranes at 42ºC for 30 minutes with a minimum of 0.5ml/cm2 of 1-0.1xSSC, 0.1% SDS, with continuous agitation. Replace the wash solution once.
  • Remove the blot with forceps and shake off excess wash solution. Blots can then be used directly for chemiluminescent detection (EZ-ECL, Cat. No. 20-500-120) of hybridized DNA, or can be stored air-dried for later detection using colorimetric detection systems.

References:

  • Sambrook, J. Fritsch, E.F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)
  • Wahl, G.M., Berger, S.L. & Kimmel, A.R. (1987) Molecular Hybridization of immobilized nucleic acids: Theoretical concepts and practical considerations. Methods Enzymol. 152:399-407.