DNA实验

Standard Ligation

2024-09-28 DNA实验加入收藏

Standard Ligation关键词： Standard Ligation来源：互联网 Prepare a ligation mix: Ligatio

Standard Ligation

关键词： Standard Ligation来源：互联网

Prepare a ligation mix:

Ligation Mix (2x)


10x ligase buffer	1.0 ml
digested vector (0.1 mg/ml)	1.0 ml
H2 O	6.0 ml
total	8.0 ml

divide ligation mix into two Eppendorf tubes

Ligation Rxn

	ｉｎｓｅｒｔ	Control
ligation mix	4.0 ml	4.0 ml
ｉｎｓｅｒｔ	1.0 ml	--- ml
T4 DNA ligase (400 u/ml)	0.2 ml	0.2 ml
Total	5.2 ml	4.2 ml

incubate for 2-3 h (or ovn) at 14℃. proceed with the transformation of the appropriate E. coli strain .

Solutions: 10x Ligation Buffer: 0.5 M Tris-HCl pH 7.8, 50 mM MgCl2 , 0.1 M b-mercaptoethanol, 50 mM ATP, 5 mg/ml BSA

Ligation Buffer (10x)


1 M Tris-HCl pH 7.8	500.0 ml
1 M MgCl2	50.0 ml
b-mercaptoethanol	7.0 ml
100 mM ATP	50.0 ml
0.1 g/ml BSA	50.0 ml
H2 O	343.0 ml
Total	1 ml

Remarks:

As usually when pipetting enzyme reactions keep the tubes on ice during all manipulations. Cip-treat a digested vector cut with only a single enzyme. If the vector was double-digested: phenolize prior to ligating. Use equimolar amounts of vector and ｉｎｓｅｒｔ. As a rule we use 50 ng vector (3 kb) and 50 ng ｉｎｓｅｒｔ (3 kb). More values are given in the table below. Recalculate accordingly when using different-length vectors. Don't use less than 8 ng or so for fragments smaller than 0.5 kb. In the case of small fragments circularization occurs and removes the F from the rxn.