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Creation and use of your infectious vector

          Stewart Laboratory Standard Operating Procedure               


TRF Southern Protocol

  1. Digest HMW DNA with Hinf1/Rsa1. To ensure complete digest start with 10 μg of DNA. Please note, if you believe that SV40 is in your telomeres you o use these enzymes.
  • 10 μg DNA
  • 10 μl NEB buffer 2
  • 10 μl Rsa1
  • 10 μl Hinf1
  • ddH2O to 100 μl
    1. Add 12 μl 5 M NaCl and 300 μl 100% EtOH and cool to -20C.
    2. 3. Spin down DNA for 15 at 4C
    3. 4. Remove supernatant and allow to air dry (do NOT over dry)
    4. 5. Resuspend in 16 μl dd H2O and OD at 1:100
    5. 6. Pour a 0.5% agarose gel with 0.5X TBE containing 0.5 μg/ml EtBr.
    6. 7. Label a ladder with g32PATP
  • 1 μg HMW DNA ladder
  • 2 μl 10X kinase buffer
  • 2.5 μl g32P ATP (3000 Ci/mmol)
  • 1 μl T4 kinase
  • dd H2O to 20 μl
  • incubate for 30 at 37C
  • clean-up with a microspin column S200 and count in scintillation counter. You want to use between 1 and 5 μl of this and you want it to be at approximately 0.5-1 x 106 CPM/μl.
    1. Load equal amounts of DNA on gel and the labeled ladder.
    2. Run gel at about 50 volts for approximately 24 hours for the best resolution (assuming you are using a large gel rig).
    3. Remove gel from rig and take a picture with a ruler. This gives you a sense of how well the digest went (you should see a smear) and whether things are evenly loaded.
    4. Place 2 pieces of Whatman paper under gel and saran wrap on top. Dry gel for approximately 45-60 at 63C. DO NOT completely dry the gel, it should remain wet!
    5. Denature the gel for 15 in 1000 ml:
    1. Neutralize the gel for 10 to 30 in 1000 ml:
    1. Remove gel and place in 50 ml hybridization buffer:
    1. Add 20-50 μl hot probe labeled as follows:
  • 5 μl @ 20 μM (C3TA2) 4
  • 60 μl dd H2O
  • 10 μl T4 kinase 10X buffer
  • 20 μl g32P ATP (3000 Ci/mmol)
  • 5 μl T4 Kinase
  • Incubate at 37C for 35 and clean-up through a G25 column
    1. Incubate at 37C overnight
    2. Wash gel:
  • 4X SSC, 0.1% SDS @ room temp for 10
  • 4X SSC, 0.1% SDS @ 55C for 10
  • 2X SSC, 0.1% SDS @ 55C for 30
    1. Check before third wash as not to over wash.
    2. Expose to film.