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Gd Andysis
PAGE
Cleaning and assembling the glass plate sandwich
The glass plates were cleaned first with distilled water and alcohol The IPC (Precision
Castor) flat was placed on the bench wth glass plate fac~ng upward The front glass plate
was placed onto the IPC and spacers with the siliconized or coated surface facing down
The clamps were slided over the IPC assembly The glass plate was nnsed with cold Bind
silane (Amersham, U S A) and IPC was rinsed with repel silane (Amersham, U S A)
Gel Casting
The gel was cast with the glass plate assembly in the horizontal position Two full-length
clamps secure the assembly and allow attachm~i~t of the preclslon caster base to the
bottom of the glass plate sandwich The gel matrix was prepared using 5% acrylarnide,
0 25% bisacryl, and 7 5 M urea In 50 rnM TndSO mM bonc ac~d/lmM EDTA Sambrook
et al, (1989) The gel solution was degassed for 5-1 5 min under a strong vacuum (5 26
in Mg) to insure reproducible gel poroslty to remove dissolved oxygen Afler degassing
450 p1 of 10% Ammomum persulphate (APS) (100 mglml) and 100 pl of
retramethylenediamine (TEMED) was added to 100 rnl of gel solut~on and gel was cast
The gel matrix was injected from the bottom of glass plate sandwich Flat edge of comb
was inserted not more than 5 rnm past the short glas- plate After the gel was cast the
assembly was leveled for polymerization
The gel was pre-run in 0 5 x TBE, freshly prepared from a 10 x TBE (100 mM Tnd 100-
mM boric acid12 mM EDTA) stock solution Once the solution redches the top of glass