STEWART LABORATORY

 

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TRF SOUTHERN PROTOCOL

 

 

 

DIGEST HMW DNA WITH Hinf1/Rsa1

 

We typically digest over night. To ensure complete digest start with 10 μg of DNA (Note, if you believe that SV40 is in your telomeres you should not use these enzymes)

      Mix:

 

         10 μg DNA

 

         10 μL NEB buffer 2

 

         10 μL Rsa1

 

         10 μL Hinf1

 

         ddH2O to 100 μL

 

      Add 12 μL 5 M NaCl and 300 μL 100% EtOH and cool to -20C

 

      Spin down DNA for 15 min at 4C

 

      Remove supernatant and allow to air dry (do NOT over dry)

 

      Re-suspend in 16 μL ddH2O and OD at 1:100

 

      Pour a 0.5% agarose gel with 0.5X TBE containing 0.5 μg/mL EtBr

 

 

LABEL A LADDER WITH γ-32P-ATP

 

      Mix:

 

         1 μg HMW DNA ladder

 

         2 μL 10X kinase buffer

 

         2.5 μL γ-32P-ATP (3000 Ci/mmol)

 

         1 μL T4 kinase

 

         ddH2O to 20 μL

 

      Incubate for 30 min at 37C

 

      Clean-up with a microspin column S200 and count in scintillation counter. You want to use 1-5 μL of this and you want it to be at approximately 0.5-1.0 x 106 CPM/μL

 

 

GEL RUN

 

      Load equal amounts of DNA on gel and the labeled ladder

 

      Run gel at about 50 volts for approximately 24 hours for the best resolution (assuming you are using a large gel rig)

 

      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

 

      Place 2 pieces of Whatman paper under gel and saran wrap on top. Dry gel for approximately 45-60 min at 63C. DO NOT completely dry the gel, it should remain wet!

 

 

GEL TREATMENT AND PROBE

 

      Denature the gel for 15 min in 1000 mL:

 

         300 mL 5M NaCl (1.5 M)

 

         100 mL 5M NaOH (0.5M)

 

         600 mL H2O

 

 

      Neutralize the gel for 10-30 min in 1000 mL:

 

         300 mL 5M NaCl (1.5 M)

 

         250 mL 2.5 ml Tris pH 8 (0.625 M)

 

         450 mL H2O

 

 

      Remove gel and place in 50 mL hybridization buffer:

 

        

50 mL P wash

13.4 g NaH2PO4

1.2 g NaPyrophosphate

 
25 mL 20X SSC

 

         1 mL P wash*

 

         10 mL 50X Denharts

 

         2 mL 10% SDS

 

         H2O to 100 mL

 

 

      Add 20-50 μL hot probe labeled as follows:

 

         Mix:

 

        5 μL of 20 μM (C3TA2)4

 

        60 μL ddH2O

 

        10 μL T4 kinase 10X buffer

 

        20 μL γ-32P-ATP (3000 Ci/mmol)

 

        5 μL T4 Kinase

 

         Incubate at 37C for 30 min and clean-up through a G25 column

 

 

INCUBATE AND WASH

 

      Incubate at 37C overnight

 

      Wash gel:

 

         4X SSC, 0.1% SDS @ room temp for 10 min

 

         4X SSC, 0.1% SDS @ 55C for 10 min

 

Do not overwash. The 3rd wash may not be needed

 

         2X SSC, 0.1% SDS @ 55C for 30 min

 

 

EXPOSE TO FILM