110
GUDRUN A. RAPPOLD, KARIN RIED, ALBRECHT KLINK, ERCOLE RAO AND BIRGIT WEISS
are sensitive to CpG methylation, e.g., N otl, Bssll, Sstll or Eagi. Enzymes
with recognition sequences containing also A and T's also cleave outside
CpG islands, e.g., Sali, Mlul, Nrul and Clal (Bickmore and Bird 1993).
Subprotocol 4
Gel Electrophoresis
Materials
Reagents
• Agarose
• LMP-agarose (low melting point agarose)
• EtBr ( ethidium bromide)
Solutions
• TBE (10xTBEis 108gTris base, 55 gboricacidand40ml0.5MEDTA (pH
7,5) in 1 1)
• Denaturing solution (0,5 M NaOH + 1.5 M NaCl)
• Neutralization solution (50 mM Na phosphate (pH 6.5))
Supplies
• Gel tray, frame and comb
• Scalpels
• Syringe and needle
ln advance
Pre-cool1-31 of 0.25 x TBE in the apparatus to 10°C, 1 or 2 h before gel run
Time required
• Day 4: 1-2 h
• Day 6: 3 h
Procedure
1. Melt 0.8% agarose in 0.25 x TBE and cool to 50-60°C before pouring into
the frame. Insert comb immediately.
2. Remove comb carefully and load the DNA block by using 2 sterile scalpels. Flame (and cool) scalpel between samples if you have digested
blocks with different enzymes. Load DNA marker on both sides of
the gel.
GUDRUN A. RAPPOLD, KARIN RIED, ALBRECHT KLINK, ERCOLE RAO AND BIRGIT WEISS
are sensitive to CpG methylation, e.g., N otl, Bssll, Sstll or Eagi. Enzymes
with recognition sequences containing also A and T's also cleave outside
CpG islands, e.g., Sali, Mlul, Nrul and Clal (Bickmore and Bird 1993).
Subprotocol 4
Gel Electrophoresis
Materials
Reagents
• Agarose
• LMP-agarose (low melting point agarose)
• EtBr ( ethidium bromide)
Solutions
• TBE (10xTBEis 108gTris base, 55 gboricacidand40ml0.5MEDTA (pH
7,5) in 1 1)
• Denaturing solution (0,5 M NaOH + 1.5 M NaCl)
• Neutralization solution (50 mM Na phosphate (pH 6.5))
Supplies
• Gel tray, frame and comb
• Scalpels
• Syringe and needle
ln advance
Pre-cool1-31 of 0.25 x TBE in the apparatus to 10°C, 1 or 2 h before gel run
Time required
• Day 4: 1-2 h
• Day 6: 3 h
Procedure
1. Melt 0.8% agarose in 0.25 x TBE and cool to 50-60°C before pouring into
the frame. Insert comb immediately.
2. Remove comb carefully and load the DNA block by using 2 sterile scalpels. Flame (and cool) scalpel between samples if you have digested
blocks with different enzymes. Load DNA marker on both sides of
the gel.