30.DNA 塩基配列決定法（RI法）（坂中千恵）
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
ssDNA:1~3ｵg/1reaction
dsDNA:5~10ｵg/1reaction
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
4)reaction
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
5)loading
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。
反応後のサンプル（4）のあと）は-20℃で保存が可能である。