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analysis of genomic DNA by southern blotting

来源:生物谷  2007/7/5 22:41:00   访问量:6331
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Reagent Final Conc. Volume Mass Salmon Testes DNA (Sigma D-9156),
10.4 mg/ml, boil 5 min. before use optional: dextrane sulfate dH2O ad 20 ml

(1 hour)

  1. Isolate 1-10 µg target cDNA from vector with restriction digestion, agarose gel electrophoresis, gel extraction of appropriate band. Resuspend in 10 mM Tris.

  2. Label 25-50 ng cDNA-probe with 32P using the Prime It® II Random Primer Labeling Kit (Stratagene #300385).

    Supplies & Equipment:

    • Prime It® II Random Primer Labeling Kit (Stratagene #300385)
    • Waterbath 37°C
    • Waterbath 100°C
    • Microfuge

    Reagents:

    • Random 9-mer primers or specific primers from PCR (10 µM) or sequencing reaction (10 ng/µl)
    • 5 x *dATP buffer (for use with [a-32P]dATP) containing dGTP, dCTP, dTTP, or 5 x *dCTP buffer (for use with [a-32P]dCTP) containing dGTP, dATP, dTTP
    • [a-32P]dATP) or [a-32P]dCTP) - not provided by the kit
    • Exo(-) Klenow fragment of DNA polymerase I (5 U/µl)
    • Stop mix (0.5M EDTA, pH 8.0)

    Procedure:

    1. Add to the bottom of a microfuge tube:

      • 25-50 ng (1-23 µl) of template DNA
      • 0-23 µl dH2O
      • 10 µl primer (either random oligonucleotide primers from kit or specific primers (diluted: 10 µM or 10 ng / µl) from PCR or sequencing)
    2. Boil reaction tube in water bath for 5 minutes, then centrifuge briefly at room temperature to collect condensed liquid from cap of tube.

    3. Add the following reagents to the reaction tube:

      • 10 µl of 5 x primer buffer (either 5 x *dATP or 5 x *dCTP buffer)
      • 5 µl of labeled nucleotide (either [a-32P]dATP) or [a-32P]dCTP)
      • 1 µl Exo(-) Klenow enzyme (5 U/µl)

      Mix the reaction components thoroughly with pipet tip.

    4. Incubate the reaction at 37 - 40°C for 2 - 10 minutes.
    5. Stop the reaction by adding 2 µl of stop mix.
    6. Purify probe with either one of the following kits:
      • NucTrap® Probe Purification Push Columns (Stratagene #400701), for DNA- or RNA-probes 17 bp - 50 kb
      • Qiaquick PCR Purification Kit (Qiagen #2804) for DNA-probes 100 bp - 10 kb
    7. Count radioactivity of probe. Take 1 µl probe, dilute with appropriate amount of scintillation fluid, use same amount of scintillation fluid as negative control. Count. You should have 1-5 x 106 cpm per ml hybridization reaction (20-100 x 106 cpm).
    8. Denature probe by boiling 5 min. and chill on ice immediately before use.

    Time Required: 30 minutes 

(overnight)

  1. Add denatured probe to prehybridization solution.
    (If Denhardt's was in the prehybridization solution and should not be in the hybridization solution, prepare fresh prehybridization solution without Denhardt's for hybridization.)
    Incubate overnight (12-24 hours) at 42°C (or at 68°C without formamide) in hybridization oven.
    The hybridization solution can be reused.
    (Pour into a 50 ml plastic disposable centrifuge tube. The probe is good for a couple of weeks, and must be boiled 5 min and chilled on ice before reuse.)

(2 hours)

  1. Soak membrane twice for at least 15 minutes each with 100 ml of 7 x SSPE / 0.1-0.5% SDS at room temperature. Perform this step in hybridization oven rotating at very low speed.
    (for 500 ml: 175 ml 20 x SSPE, 2,5 - 12,5 ml 20% SDS, 322.5 - 312.5 ml dH2O)

  2. Then soak the membrane twice for at least 15 minutes each in 100 ml of 1 x SSPE / 0.5-1% SDS at 37°C. Perform this step in hybridization oven rotating at very low speed.
    (for 500 ml: 25 ml 20 x SSPE, 12.5 - 25 ml 20% SDS, 462.5 - 450 ml dH2O)

  3. Finally soak for 1 hour in 100 ml of 0.1 x SSPE / 1% SDS at 68°C.
    (for 500 ml: 2.5 ml 20 x SSPE, 25 ml 20% SDS, 472.5 ml dH2O)
    For detection of poorly matched hybrids use a lower temperature in the last wash. Check radioactivity with Geiger-counter. Do not expect a signal from single copy genes.

(1 to 6 days)

After washing, blot the membrane with filter paper (Whatman 3MM or S&S GB003) to remove most of the excess moisture. Wrap moist blots in plastic wrap prior to autoradiography. Expose to X-ray film with an intensifying screen at -80°C for 1 to 6 days.

(1 hour)

Do not allow blot to dry at any time prior to removing the probe, as drying will cause the probe to bind irreversibly. To remove probe from blot for reuse. incubate one of the following solutions:

  • Wash blots in 50% formamide, 6 x SSPE for 30 minutes at 65°C.
    (
    for 50 ml: 25 ml deionized formamide, 15 ml 20 x SSPE, 10 ml dH2O)
    Rinse in 2 x SSPE, remove excess liquid by dabbing the blot on 3MM paper. Wrap in Saran wrap and store. Expose overnight to check for the absence of radioactivity. After stripping probe, start again with prehybridizing step.

 

  • Microfuge
  • Waterbath 37, 100°C
  • Agarose gel electrophoresis supply
  • S&S Nytran Membranes
  • Whatman 3MM paper
  • paper towels
  • UV-crosslinker
  • hybridization bottles
  • hybridization oven at 42, 37, 68°C
  • Pyrex glass trays

  • Salmon Testes DNA (Sigma D-9156), 10.4 mg/ml
  • 1 % Ethidium bromide stock solution (10 µg/µl)
  • Agarose
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