Embryo Lysis Buffer (refrigerate)
Final concentrations: in 500ml
| for 500 ml: | |
|---|---|
| 50 mM KCl | 25 ml 1M KCl |
| 10 mM Tris pH 8.3 | 10 ml 1M Tris pH 8.5 |
| 2 mM MgCl2 | 1 M MgCl2 |
| 0.1 mg/ml gelatin | 5 ml 10 mg/ml gelatin |
| 0.45% NP-40 | 2.25 ml NP-40 |
| 0.45% Tween-20 | 2.25 ml Tween-20 |
| 0.1 mg/ml Proteinase K | 454.5 ml H2O with 7 µl Proteinase K per ml |
Lyse embryo tissue 1 hr to O/N (depending on size of tissue) at 55°C in 500 µl Embryo Lysis Buffer.
Boil tubes for 10min.
Use 1 µl in PCR.
Alternative method for preparing embryo tissue for PCR (Adult Mouse Tissue Preparation for PCR)
Lysis Buffer:
0.1M Tris pH 8.5
5 mM EDTA
0.2% SDS
0.2 M NaCl
Add 7µl Proteinase K per ml
Add 500 µl Lysis Buffer to tissue in tube.
Incubate overnight at 55°C with agitation.
Vortex tubes briefly, pellet cell debris (14,000 rpm, 10 min).
Pour supernatant into a fresh, labeled tube (discard tube with cell debris pellet)
Add 500 µl isopropanol, invert several times to precipitate DNA.
Incubate at room temp ~20 min.
Pellet DNA, aspirate supernatant, dry pellet in speedvac.
Resuspend DNA pellet in 500 µl water.
Incubate overnight at 55°C with agitation.
Use 1 µl DNA in PCR, store samples at 4°C.
PCR Master Mix:
1 µl template DNA
0.5 µl 3' primer (100 µM)
0.5 µl 5' primer (100 µM)
1µl dNTPs (10 mM)
5 µl PCR Buffer (10x)
5 µl MgCl2 (25 mM)
0.25 µl Taq (5U/µl)
36.75 µl H2O
Total: 50 µl
Run samples on 1.5% high resolution agarose (AMRESCO) gel with 100bp ladder.