Standard Ligation

2010-03-05 22:39 · Dave

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 di

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.

F Used in Standard Ligation

vector (50 ng, 3kb)

Length of F

(kb)

Amount of F (ng)

0.5

8.3

1

16.7

1.5

25

2

33.3

2.5

41.7

3

50

3.5

58

4

66.7

5

83.3

6

100

7

116.3

Materials:

Reagent/Tool

Supplier

Cat.-#

BSA

ATP

T4 DNA Ligase

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