Tail and Yolk Digests for PCR
Tail Digests
- 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.
- Extract with 500 µl P:C 1:1, vortex and spin 2 minutes at high speed. Transfer 600 µl aqueous to new tube.
- Add 550 µl isopropanol, vortex and let stand 2-5 minutes to precipitate DNA.
- Spin at high speed 5 minutes, remove liquid and wash pellet with 2X 70% ethanol. Dry pellet at RT for ~1hour.
- Resuspend in 100 µl TE or H2O. It may take O/N at 4°C to resuspend.
- 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
- Put yolk sac in 50 µl PBT/0.2% Tween® 20 with 1 µl fresh 10 mg/ml proteinase K.
- Incubate 55°C for 3 hours to O/N.
- Boil samples for 5 minutes to inactivate proteinase K.
- Spin briefly to collect pellet cell debris at bottom of tube.
- 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.
- Put yolk sac in 50 µl ES cell DNA digest buffer and digest O/N at 55-60°C.
- Boil samples for 5 minutes to inactivate proteinase K.
- Precipitate DNA by adding 100 µl of ice-cold ethanol + 15 µl 5M NaCl. Keep on ice for 15 minutes.
- Spin down 4°C for 15 minutes.
- Wash pellet with 70% ethanol.
- 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.