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)

           

Ø      Mix:

 

§         10 μg DNA

 

§         10 μL NEB buffer 2

 

§         10 μL Rsa1

 

§         10 μL Hinf1

 

§         ddH2O to 100 μL

 

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

 

Ø      Spin down DNA for 15 min at 4°C

 

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

 

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

 

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

 

 

LABEL A LADDER WITH γ-32P-ATP

 

Ø      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

 

Ø      Incubate for 30 min at 37°C

 

Ø      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

 

Ø      Load equal amounts of DNA on gel and the labeled ladder

 

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

 

Ø      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

 

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

 

 

GEL TREATMENT AND PROBE

 

Ø      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

 

 

Ø      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

 

 

Ø      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

 

 

Ø      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 37°C for 30 min and clean-up through a G25 column

 

 

INCUBATE AND WASH

 

Ø      Incubate at 37°C overnight

 

Ø      Wash gel:

 

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

 

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

 

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

 

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

 

 

EXPOSE TO FILM