Skip to Content

Tail and Yolk Digests for PCR

Tail Digests

  1. Cut tails or toes into 2-3 small pieces and add 750 µl tail digest buffer containing 0.2 mg/ml proteinase K. Heat 55°C for 3 hours to O/N. If rocking, vortex briefly after 1 hour and return to 55°C.
  2. Extract with 500 µl P:C 1:1, vortex and spin 2 minutes at high speed. Transfer 600 µl aqueous to new tube.
  3. Add 550 µl isopropanol, vortex and let stand 2-5 minutes to precipitate DNA.
  4. Spin at high speed 5 minutes, remove liquid and wash pellet with 2X 70% ethanol. Dry pellet at RT for ~1hour.
  5. Resuspend in 100 µl TE or H2O. It may take O/N at 4°C to resuspend.
  6. Use 1 µl for PCR genotyping.

Tail Digest Buffer Stock Solution

Final

  • 10mM Tris, pH 8.0
  • 25mM EDTA, pH 8.0
  • 100mM NaCl
  • 1% SDS
  • 0.2 mg/ml Proteinase K

    Add proteinase K right before use

For 100 ml

  • 1.0 ml 1.0M Tris, pH 8.0
  • 5.0 ml 0.5M EDTA, pH 8.0
  • 2.0 ml 5.0M NaCl
  • 10 ml 10% SDS
  • 10 l/ml 20 mg/ml Proteinase K

    Add proteinase K right before use

Yolk Sac Digest for PCR Genotyping

E8.5-E11.5

  1. Put yolk sac in 50 µl PBT/0.2% Tween® 20 with 1 µl fresh 10 mg/ml proteinase K.
  2. Incubate 55°C for 3 hours to O/N.
  3. Boil samples for 5 minutes to inactivate proteinase K.
  4. Spin briefly to collect pellet cell debris at bottom of tube.
  5. Take 1 µl for PCR.

For E12.5-15.5, only use part of amniotic sac.

Alternative Yolk Sac Digest for PCR Genotyping

E8.5-E9.5

Based on ES DNA prep, this has worked really well with embryo yolk sacs.

  1. Put yolk sac in 50 µl ES cell DNA digest buffer and digest O/N at 55-60°C.
  2. Boil samples for 5 minutes to inactivate proteinase K.
  3. Precipitate DNA by adding 100 µl of ice-cold ethanol + 15 µl 5M NaCl. Keep on ice for 15 minutes.
  4. Spin down 4°C for 15 minutes.
  5. Wash pellet with 70% ethanol.
  6. Resuspend in 25 µl H2O.

ES Cell DNA Digest Buffer

  • 10mM Tris, pH 7.5
  • 10mM EDTA
  • 10mM NaCl
  • 0.5% Sarkosyl

Generally, 1/10-µl dilution of DNA for PCR is used.

On those for which this doesn't work, increase the concentration to a full 1 µl per 25-µl PCR reaction.


© 2014 The University of Texas MD Anderson Cancer Center