Preparation of nuclear extracts from mouse embryos

1. Harvest embryos in ice-cold PBS.
2. Wash once in ice-cold PBS and then buffer A.
3. Cut embryos into parts and swell them in one packed embryo volume of buffer A for 15 min on ice.
4. Break the cells by rapidly pushing through a large needle, followed by a 25-gauge needle.
5. Spin the cell homogenate at 13k rpm for 20 sec
6. Resuspend the crude nuclear pellet in 2/3 of one packed cell volume of buffer C.
7. Keep on ice with rocking for 30 min
8. Spin for 5 min at 4oC.
9. The supernatant (nuclear extract) is dialyzed against buffer D for 2 hr at 4oC.
10. The dialyzed extract can be used directly for transcription or splicing assays, or quick-frozen in liquid nitrogen and stored at -70oC in aliquots.

Buffer A

 10 mM Hepes-KOH pH7.9  0.2 ml (0.5 M)
 1.5 mM MgCl2  15 ul (1 M)
 10 mM KCl  0.1 ml (1 M)
 0.5 mM DTT  5 ul (1 M)
 0.5 mM PMSF  50 ul (0.1 M)
 leupeptin  20 ul (0.5mg/ml)
 H2O  9.61 ml

Buffer C

 20 mM Hepes-KOH pH7.9  0.4 ml (0.5 M)
 1.5 mM MgCl2  15 ul (1 M)
 420 mM NaCl  1.05 ml (4 M)
 0.2 mM EDTA  4 ul (0.5 M)
 25% glycerol  2.5 ml (100%)
 0.5 mM DTT  5 ul (1 M)
 0.5 mM PMSF  50 ul (0.1 M)
 leupeptin  20 ul (0.5 mg/ml)
 H2O  5.96 ml

Buffer D




     20 mM Hepes-KOH pH7.9
     4 ml (0.5 M)

     0.1 M KCl
     10 ml (1 M)

     0.2 mM EDTA
      4 ul (0.5 M)
 


     20% glycerol
     20 ml (100%)

     0.5 mM DTT
     50 ul (1 M)

     0.5 mM PMSF
     500 ul (0.1 M)
 


     leupeptin
     200 ul (0.5 mg/ml)

     H2O
     65.21 ml


Ref: Lee et al (1988) Gene Anal Techn 5:22-31
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