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Double Digestion용 추천 Universal Buffer

Double digestion buffer 선택가이드

두 종의 제한효소를 동시에 사용하여 DNA를 절단하는 double digestion 실험은 시간을 절약하기 위해 종종 수행된다. 당사에서 판매하는 제한효소에는 universal buffer가 포함되어 있지만 (각 buffer에 따른 상대 활성은 ‘Universal buffer별 활성’ 페이지 참조), 이 중 일부 double digestion의 경우, 두 효소에 모두 적절한 buffer를 선택하는 것이 어려울 수 있다. 각기 다른 두 효소의 높은 활성은 유지하고 star activity를 방지하기 위해 적절한 buffer를 선택하는 것이 중요하다.
일반적인 double digestion에 적절한 buffer, 희석배수 및 필수 첨가시약을 아래의 표에서 확인할 수 있다.
L, M, H, T, K는 buffer 유형을 나타내고, 권장되는 최종 buffer 농도로 표시된다 (universal buffer는 10X 농도로 제공됨). BSA도 10X 농도로 공급되므로, 10배 희석하여 최종 농도 0.01%로 사용한다.
단, 아래의 표에서 확인되지 않는 조합의 경우, double digestion을 대신 각 제한효소의 조건에 따라 따로 처리할 것을 권장한다.

Enzyme	Acc I	BamH I	Bgl II	Cla I	EcoR I	EcoR V	Hinc II	Hind III	Kpn I	Nco I	Nde I
Supplied Buffer	10X M	10X K	10X H	10X M	10X H	10X H	10X M	10X M	10X L	10X K + BSA	10X H
Acc I	-	0.5X K	1X T	1X M	1X M	0.5X K	1X M	1X M	1X M	1X M + BSA	1X T
BamH I	0.5 × K	-	1X K	1X K	1X K	1X K	0.5X K	1X K	0.5X K	1X K + BSA	1X K
Bgl II	1X T	1X K	-	1X H	1X H	1X H	2X K	1X K	1X T	1X K + BSA	1X H
Cla I	1X M	1X K	1X H	-	1X H	1X H	1X M	1X M	1X M	1X K + BSA	1X H
EcoR I	1X M	1X K	1X H	1X H	-	1X H	1X M	1X M	1X M	1X K + BSA	1X H
EcoR V	0.5X K	1X K	1X H	1X H	1X H	-	2X T	1X K	0.5X K	1X K + BSA	1X H
Hinc II	1X M	0.5X K	2X K	1X M	1X M	2X T	-	1X M	1X M	1X M + BSA	1X T
Hind III	1X M	1X K	1X K	1X M	1X M	1X K	1X M	-	1X M	1X K + BSA	1X K
Kpn I	1X M	0.5X K	1X T	1X M	1X M	0.5X K	1X M	1X M	-	0.5X K + BSA	1X T
Nco I	1X M + BSA	1X K + BSA	1X K + BSA	1X K + BSA	1X K + BSA	1X K + BSA	1X M + BSA	1X K + BSA	0.5X K + BSA	-	1X K + BSA
Nde I	1X T	1X K	1X H	1X H	1X H	1X H	1X T	1X K	1X T	1X K + BSA	-
Not I	0.5X K + BSA	0.5X K + BSA	1X H + BSA	1X H + BSA	1X H + BSA	1X H + BSA	0.5X K + BSA	0.5X K + BSA	0.5X K + BSA	0.5X K + BSA	1X H + BSA
Pst I	1X M	1X K	1X H	1X H	1X H	1X H	1X M	1X M	1X M	1X K + BSA	1X H
Pvu I	0.5X K	1X K	1X K	1X K	1X K	1X K	0.5X K	1X K	0.5X K	1X K + BSA	1X K
Sac I	1X M	0.5X K	0.5X K	1X M	1X M	0.5X K	1X M	1X M	1X L	0.5X K + BSA	1X T
Sal I	1.5X T	1.5X T	1X H	1X H	1X H	1X H	1.5X K	1.5X K	1.5X T + BSA	1.5X T + BSA	1X H
Sma I	1X T + BSA	0.5X T + BSA	1X T + BSA	1X T + BSA	1X T + BSA	0.5X K + BSA	1X T + BSA	1X T + BSA	1X T + BSA	1X T + BSA	1X T + BSA
Spe I	1X M	1X K	1X H	1X M	1X H	1X H	1X M	1X M	1X M	1X K + BSA	1X H
Sph I	0.5X K	1X K	1X H	1X H	1X H	1X H	2X T	1X K	0.5X K	1X K + BSA	1X H
Xba I	1X M	0.5X K	2X T	1X M	1X M	2X T	1X M	1X M	1X M	1X M + BSA	1X T
Xho I	1X M	1X K	1X H	1X H	1X H	1X H	1X M	1X M	1X M	1X K + BSA	1X H

Enzyme	Not I	Pst I	Pvu I	Sac I	Sal I	Sma I	Spe I	Sph I	Xba I	Xho I
Supplied Buffer	10X H + BSA + Triton	1X H	10×K + BSA	10X L	10X H	10X T + BSA	10X M	10X H	10X M + BSA	10 X H
Acc I	0.5X K + BSA	1X M	0.5X K	1X M	1.5X T	1X T + BSA	1X M	0.5X K	1X M	1X M
BamH I	0.5X K + BSA	1X K	1X K	0.5X K	1.5X T	0.5X T + BSA	1X K	1X K	0.5X K	1X K
Bgl II	1X H + BSA	1X H	1X K	0.5X K	1X H	1X T + BSA	1X H	1X H	2X T	1X H
Cla I	1X H +BSA	1X H	1X K	1X M	1X H	1X T +BSA	1X M	1X H	1X H	1X H
EcoR I	1X H + BSA	1X H	1X K	1X M	1X H	1X T + BSA	1X H	1X H	1X M	1X H
EcoR V	1X H + BSA	1X H	1X K	0.5X K	1X H	0.5X K + BSA	1X H	1X H	2X T	1X H
Hinc II	0.5X K + BSA	1X M	0.5X K	1X M	1.5X K	1X T + BSA	1X M	2X T	1X M	1X M
Hind III	0.5X K + BSA	1X M	1X K	1X M	1.5X K	1X T + BSA	1X M	1X K	1X M	1X M
Kpn I	0.5X K +BSA	1X M	0.5X K	1X L	1.5X T +BSA	1X T +BSA	1X M	0.5X K	1X M	1X M
Nco I	0.5X K + BSA	1X K + BSA	1X K + BSA	0.5X K + BSA	1.5X T + BSA	1X T + BSA	1X K + BSA	1X K + BSA	1X M + BSA	1X K +BSA
Nde I	1X H + BSA	1X H	1X K	1X T	1X T + BSA	1X H	1X H	1X T	1X H	1X H
Not I	-	1X H + BSA	2X K + BSA	0.5X K + BSA	1X H + BSA	0.5X T + BSA	1X H + BSA	1X H + BSA	0.5X K + BSA	1X H +BSA
Pst I	1X H + BSA	-	1X K	1X M	1X H	0.5X T + BSA	1X H	1X H	1X M	1X H
Pvu I	2X K + BSA	1X K	-	0.5X K	1.5X K + BSA	1X K + BSA	1X K	1X K	0.5X K	1X K
Sac I	0.5X K + BSA	1X M	0.5X K	-	1.5X T + BSA	1X T + BSA	1X M	0.5X K	1X M	1X M
Sal I	1X H + BSA	1X H	1.5X K + BSA	1.5X T + BSA	-	1.5X T + BSA	1X H	1X H	1.5X T	1X H
Sma I	0.5X T + BSA	0.5X T + BSA	1X K + BSA	1X T + BSA	1.5X T + BSA	-	1X T + BSA	0.5X T + BSA	1X T + BSA	1X T +BSA
Spe I	1X H + BSA	1X H	1X K	1X M	1X H	1X T + BSA	-	1X H	1X M	1X H
Sph I	1X H + BSA	1X H	1X K	0.5X K	1X H	0.5X T + BSA	1X H	-	2X T	1X H
Xba I	0.5X K + BSA	1X M	0.5X K	1X M	1.5X T	1X T + BSA	1X M	2X T	-	1X M
Xho I	1X H + BSA	1X H	1X K	1X M	1X H	1X T + BSA	1X H	1X H	1X M	-

Notes:
각 효소 10 unit은 50 ㎕ 반응액에서 37℃, 1 시간 내에 1 ㎍의 DNA를 완전히 분해한다.
Star activity를 최소화하려면 glycerol 농도가 10% 미만이어야 한다.
고차 구조의 DNA는 두 제한효소의 인식서열이 서로 가까울 경우 DNA가 완전히 절단되지 않을 수 있다.