상품 상세보기 : Cloning Enzymes

AnyFusion^®

Introduction

AnyFusion^® provides an extremely easy, efficient and rapid method for cloning. AnyFusion^® contains an optimized blend of Genenmed^®'s proprietary enzymes. AnyFusion^® utilizes a ligase-independent homologous annealing system. The insert is prepared by PCR and directly coned into the linearized vector. For general cloning, the PCR-prepared insert does not need and further purification steps to remove the primers and/or dNTPs. The single-cut cloning vectors, even without further treatments, do not make a self-ligated background, and can be used as effectively as the double-cut vectors. The cloning efficiency and accuracy is almost 100%, ensuring that analysis of one or two colonies can complete cloning. AnyFusion^® also clones multiple inserts. Therefore, AnyFusion^® really establishes a new standard in cloning.

Overview for use

AnyFusion^® Protocol

1. Vector preparation

1) Cut the vector to linearize. Single or double cuts do not make any difference in cloning results. Complete digestion is crucial.

2) Heat-inactivate the restriction enzyme(s) used if it is heat-labile. If it is heat-resistant, use a PCR clean-up kit to remove the enzyme(s)

3) Use distilled water to adjust the vector concentration. 10~100ng of the linearized vector is used for AnyFusion^® reaction.

2. Insert preparation

1) Perform PCR using Pfu polymerase (See Fig.3 for designing the primer). If the PCR template is a plasmid containing the same antibiotic resistance gene as the cloning vector, it can make a cloning background. To reduce it, pre-cutting the template vector before PCR is recommended.

2) If the PCR result shows only a specific product, no purification is needed. Otherwise, agarose gel purification is recommended. For cloning multiple inserts, purification using a PCR clean-up kit or through agarose gel electrophoresis is recommended. 10~100ng of the insert is used for AnyFusion^® reaction.

[Designing PCR primers]

1. The 14 nucleotides homologous to the cloning vector is needed. The 10 nucleotides in length shows ~80% cloning efficiency.

2. To regenerate the restriction enzyme site, include the nucleotides in ( ) in Fig.3 to the primer sequence.

3. AnyFusion^® reaction

1) Prepare the following reaction mixture:

Vector	1ul (10~100ng)
Insert	1ul (10~100ng)
10X AnyFusion^® Buffer	1ul
Distilled water	6ul
AnyFusion^®	1ul
Total	10ul

[IMPORTANT]

Vector : Insert = 1 : 1~2 (molar ratio)

Vector : InsertA : InsertB = 1 : 2 : 2 (molar ratio)

2) Incubate for 10min at 55℃.

3) Incubate for 10min in ice. During this step, thaw competent cells.

4) Transform all the reaction mixture to 40~50ul competent cells.

5) Incubate for 30min in ice.

6) Heat shock for 1min at 42℃.

7) Add 500ul LB medium and incubate for 1h at 37℃.

8) Spread all cells on the agar plate containing an antibiotic.

9) Incubate overnight at 37℃.

4. Cloning results