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GUDRUN A. RAPPOLD, KARIN RIED, ALBRECHT KLINK, ERCOLE RAO AND BIRGIT WEISS
8. Pipet mixture into precooled block formers as described above.
9. Blocksare then incubated in SCE with 10 mM DTT at 37°C for 1-2 h with
shaking.
10. Transferblocksinto proteinase buffer, add proteinase Kat 2 mglml and
incubate at 50°C for 48h.
11. Wash blocks in 50 mM EDTA/10mM Tris HCl (pH 7.5), 3 x 20 min.
12. Blocks can be stored in this mixture at 4°C or loaded directly onto the
gel without rinsing.
Subprotocol 3
Restrietion Enzyme Digestion of DNA in Agarose Blocks
Materials
Reagents Infrequently cleaving restriction enzymes
Solutions • High salt buffer (100 mM Tris-Cl (pH 7.5), 100mM MgClz, 1000 mM
NaCl - 10x conc)
• Medium salt buffer (100 mM Tris-Cl (pH 7.5), 100 mM MgC12, 10 mM
DTT, 500 mM NaCl-10x conc)
• Low salt buffer (100 mM Tris-Cl (pH 7.5),100 mM MgClz, 10 mM DTT10x conc)
• Spermidine (10 mglml in H20)
• BSA (bovine serum albumin) (10% in H20)
Supplies • Falcon tubes
• Eppendorf tubes
• Sterile plastic loops
Time required
Day 3: 2 h