1.Add 750μl Reagent B (50mM Tris [pH8],10mM EDTA,100mM NaCl,1% [w/v] SDS)and 50μl 100mg/ml proteinase K to each spleen.
2.Incubate overnight at 56ºC.
3.Gently shake to dissociate tissue.
4.transfer 200μl to eppendorf tube.
5.Add 50μl 5M sodium perchlorate.
6.Incubate 25 mins 65ºC.
7.Add 200μl phenol/chloroform.
8.Mix by inversion for about 2 mins.
9.Spin 13000rpm for 2 mins.
10.Transfer aqueous phase (top layer)to new eppendorf.
14 Add 500μl ice cold 100% ethanol.
15.Spool DNA into 500μl TE 10:0.l (if no visible DNA see below).
16 Allow to resuspend at 4ºC overnight.
If no visible DNA:
1.Spin 15 mins 13000rpm at ºC.
2.Remove liquid.
3.Add 500μl 70% ethanol.
4.Spin 5 mins 13000rpm at 4ºC.
5.Remove liquid and vac dry.
6.Add 500μl TE 10:0.1.
