1.making sequence gel
6%gel 8%
urea 42g 42g
10XTBE 10ml 10ml
40%aa/bis 20ml 15ml
water up to 100ml
add 10% ammonium persulfate 600オl
TEMED 40オl

2.sequencing reaction(Seqenase Ver.2.0)
1)Template DNA
alkaline-denaturation of dsDNA
dsDNA in 18オl of water
2N NaOH 2オl
→ incubate at room tempereture for 5min
→ add 7.5M NH4oAc 6オl
ethanol 100オl
→ at -75℃ for 5min
→ cfg. and ppt. dissolved in 7.5オl of water
2)Primer anealing
Template DNA 7.5オl
reaction mixture 2オl
primer 1オl(16~100pmol)
→ heat at 65~75℃ for 20min
slowly cool down to room tempereture
3)Labeling solution
DTT 1オl
[α-35S]dATP([α-32P]dCTP etc.) 1オl
Labeling mix 0.4オl
water 1.6オl
total 4オl for 1reaction
DNA-primer mixture(2)) 10.5オl
Labeling solution(3)) 4オl
diluted enzyme* 2オl
→ mixing and incubate at 37℃ for 5min
→ transfer 3.5オl to each(A,C,G,T) termination mix tube
2.5オlof termination mix in 1tube
→ incubate at 37℃ for 5~8min
→ add 5オl of Stop solution
reaction mixture(4))
→ boiling for 10min
→ imediately cool down on ice
→ apply 2~3オl of reaction mixture for 1 lane

付) ゲルを作る時はあたためながら(Ureaを完全に溶解させるため)行い、室温まで さましてから固める。