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Preparing Embryos for TRF Analysis

Preparing Embryos for Telomere Restriction Fragment Analysis (TRF)

  • Use 50,000 cells per plug
  • Use 1 E8.5 GCN5 null embryo per 2 agarose plugs (100 µl each)
  • Use 1 E8.5 WT embryo for 4 agarose plugs (100 µl each)
  • For Southern gel, use 4-5 null embryos and 5-6 WT embryos (10 lanes total)
  1. One WT embryo + 200 µl PBS on ice.

    In Eppendorf, use 1-ml syringe and 25g needle to make embryo "mush" on ice. Then add very hot 200 µl 2X Pulse Field Agarose (1.8%) to cells and pipet up and down to mix. Quickly pipet 100 µl per plug. Solution will congeal rapidly. Avoid pipetting bubbles into the plug.
  2. One null embryo + 100 µl PBS on ice.

    In Eppendorf, use 1-ml syringe and 25g needle to make embryo "mush" on ice. Then add very hot 100 µl 2X Pulse Field Agarose (1.8%) to cells and pipet up and down to mix. Quickly pipet 100 µl per plug. Solution will congeal rapidly. Avoid pipetting bubbles into the plug.
  3. Pour agarose plugs in small wells with bottom taped. Once set, remove tape and transfer plugs from each genotype to 15-ml tube with 5 ml lysis buffer**.
  4. Incubate all plugs at 55°C for 2 days.
  5. Wash plug 5X, 4 hours each, in 15 ml TE at 4°C to dialyze.

    You do not want to see any bubbles when you shake the plugs after your last wash. Then, you can store plugs indefinitely at 4°C.
  6. Restriction enzyme digestion.

    Digest one plug per genotype overnight at 37°C as follows:

    174 µl H2O
    20 µl 10X NEB buffer #2
    2 µl HinfI (NEB)
    2 µl RsaI (NEB)
    2 µl BSA
  7. Pulse field gel electrophoresis.

    The next day cut the plug in half, using a razor blade and ruler to make sure sizes are uniform. Save the other half of plug in 0.5X TBE at 4°C. It will keep for several months.

    Keeping gel on benchtop, fill each well with 0.5X TBE and load 1/2 plug into well of 0.8% TBE gel (1.29 g PF agarose + 160 ml 0.5X TBE) made with pulse field agarose. It is easy to load if you use a metal spatula and pipette tip.

    Seal the plug into the well using melted agarose. Leave one well open so you can load molecular weight marker after gel is set up in buffer.

  8. Run gel with pulse field electrophoresis in 0.5X TBE using BioRad FIGEMapper power supply with circulation on. Gel run = 16 hrs.
  9. Stain gel with EtBr and take picture of gel with ruler for marking molecular weight standards. Make sure all loaded lanes have DNA that is well digested.
  10. Dry down gel at 75°C for 1.5 hr on two sheets Whatman paper, with gel covered with Saran Wrap. Mark the orientation of the gel and location of marker lane on Saran Wrap. Put nothing else on gel. Gel can be stored like this in a drawer.
  11. Rehydrate gel in 2X SSC.

    Carefully remove Whatman paper from bottom of gel. Cut the corner of gel nearest the marker lane. Flip gel over with gloved hands and carefully rub off any adhering paper.

    Be careful -- gel is fragile.
  12. C-rich oligo hybridization.

    Roll gel and place in hybridization tube with ~40-50 ml prehyb for 4 hours to O/N at 58°C.
  13. Label C-rich oligo to detect G-rich single strand overhang.

    To kinase oligo:

    4 µl C-rich oligo (1 µg/µl) (5'-CCCTAACCCTAACCCTAACCC-3')
    2 µl T4 PNK forward reaction buffer
    8 µl H2O
    5 µl gamma-32P-ATP
    1 µl T4 polynucleotide kinase

    Mix oligo and water together and boil for 1 min. Place on ice for quick chill to ensure that there is no secondary structure. Then add rest of reaction. Incubate 15 minutes to 1hour at 37°C. Add 0.5mM EDTA to stop reaction.

  14. Purify oligo on G-25 spin column. Count with scintillation counter.

  15. Use 500,000 cpm/ml hyb solution (Prehyb solution** + kinased oligo). Use a minimum 5 ml hyb solution. Hybridize at 58°C O/N.
  16. Washes:

    Quick wash 4X in SSC 0.1% SDS
    3X for 20 minutes in 4X SSC 0.1% SDS, 80 mls each at RT
    3X for 20 minutes in 4X SSC 0.1% SDS, 80 mls each at 55°C
  17. Wrap gel in plastic wrap and expose on MS film for 3-5 days at -80°C.
  18. Gel can be stored in desk drawer until ready for next hybridization. Rehydrate in 2X SSC before hybridizing.
  19. G-rich oligo hybridization to detect length of double stranded telomeric DNA.

    You need to denature the DNA in the gel prior to hybridization!!!


    Denature for 1 hour at RT in 0.6M NaCl/0.2M NaOH
    Neutralize for 1 hour at RT in 1.5M NaCl/0.5M Tris, pH 7.4
    Rinse gel in H2O for 30 minutes at RT
  20. Repeat steps 12-17 with G-rich oligo (5'-GGTTAGGGTTAGGGTTAGGG-3').

**Solutions

Lysis Buffer

  • 2% N-lauryl sarkosine
  • 400mM EDTA
  • 1 mg/ml proteinase K
  • Store at 4°C

50 ml

  • 1 g
  • 40 ml 0.5M
  • 2.5 ml 20 mg/ml

Prehyb [4 L]

Heat and stir until SDS dissolves. Warm to 60°C before use.

  • 0.5M EDTA [8 ml]
  • SDS Powder [280 g]
  • 1M Sodium Phosphate, pH 7.2 [2000 ml]
  • H2O [1200 ml]

100 ml

Heat and stir until SDS dissolves. Warm to 60°C before use.

  • 0.2 ml
  • 7 g
  • 50 ml
  • 30 ml

1M Sodium Phosphate, pH 7.2

  • 1M Na2HPO4
  • 1M NaH2PO4-H2O

100 ml

  • 68.4 ml
  • 31.6 ml

© 2014 The University of Texas MD Anderson Cancer Center