AnyFusion®XL 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®XL 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®XL 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®XL reaction
1) Prepare the following reaction mixture:
|
Vector |
1ul (10~100ng) |
|
Insert |
1ul (10~100ng) |
|
10X AnyFusion®XL Buffer |
1ul |
|
Distilled water |
6ul |
|
AnyFusion®XL |
1ul |
|
Total |
10ul |
[IMPORTANT]
Vector : Insert = 1 : 1~2 (molar ratio)
Vector : InsertA : InsertB = 1 : 2 : 2 (molar ratio)
2) Incubate for 5min at 55℃.
3) Incubate for 5min 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
