2×In-Fusion Cloning Mix (for Molecular clone)

Product Information

Product Description/Introduction

2×In-Fusion Cloning Mix are designed for fast, directional cloning of one or 2~3 multiple fragments of DNA into any vector,especially suitable for single fragment. It fuses DNA fragments (e.g., PCR-generated inserts and linearized vectors) efficiently and precisely by recognizing 15-bp overlaps at their ends. These 15-bp overlaps can be engineered by designing primers for amplification of the desired sequences.

The premix does not depend on the ligase system, which greatly reduces the background of vector self-ligation, and it does not need to consider the restriction endonuclease site contained in the inserted fragment itself. For single fragment cloning into vector, the proportion of positive clones obtained as high as 99%. With the mix, all operations can be completed without thermostat equipments from DNA sample preparation to plate transformed cells within a few hours.

Storage and Shipping Conditions

Ship with wet ice; store at -20°C, valid for 12 months.

Product Contents

Assay Protocol / Procedures

Perform ligation reaction

1. To an autoclaved, 1.5-ml microcentrifuge tube, add the following(recommend 10-uL reaction system):

2. Mix gently and centrifuge briefly to bring the contents to the bottom of the tube.

3. Incubate at ice-water bath (ice-water mixture) for 5 - 10 minutes.

4. Place the tube on ice and proceed immediately to perform transformation reaction. Or you can store the ligation mixture at 20°C until you are ready.

Perform transformation reaction

5. Add appropriate ligation mixture into chemically competent cells (such as E.coli DH5α, E.coli Top10, etc.) and mix by tapping gently. Do not mix by pipetting up and down. The remaining ligation mixture(s) can be stored at 20°C.

5. Incubate for 30 minutes on ice.

6. Incubate for exactly 90 seconds in the 42°C water bath. Do not mix or shake.

7. Remove the centrifuge tubes from the 42°C bath and place them on ice for 2-5 minutes.

8. Add 900 µL of SOC or LB medium. Sterile technique must be practiced to avoid contamination. Shake the the centrifuge tube(s) at 37°C for 1 hour at 225 rpm in a shaking incubator.

9. Spread appropriate volume from each transformation centrifuge tube on separate, labeled LB agar plates. The remaining transformation mix may be stored at 4°C and plated out the next day, if desired.

10. Invert the plate(s) and incubate at 37°C overnight.

Analyze transformants

11. Select colonies and analyze by plasmid isolation, PCR, or sequencing.

Note

1. The vector DNA and insert DNA should be gel purified and analyse their quality and concentration  by electrophoresis. Water can be omitted in ligation reaction if the concentration is low.

2. The Tm value between the overlapping regions of multiple fragments should be consistent and >60.

3. It is recommended that the molar ratio of vector and insert is 1:1~1:3; when 2-3 fragments are connected, the molar ratio between each fragment is 1:1, and the ligation reaction system can be scaled up in equal proportions.

4. If the total volume of vector and insert is more than 5 μL, you may scale the ligation system to 20 μL.

5. The volume of the ligation product should not exceed 1/10 of the volume of the competent cells, otherwise the transformation efficiency will be significantly reduced. The volume of the ligation product and the competent cells can be increased in equal proportions (for example, 20 μL of ligation system transforms 200 μL of competent cells).

6. The 2×Universal Ligation Mix should be kept at -20°C until within 5-10 minutes of use and returned immediately to -20°C after use. It is recommended to freeze in aliquot to reduce freeze-thaw cycles.

7. If electroporation is used for transformation, vector DNA and insert DNA should be purified by column method or ethanol precipitation method.

For Research Use Only!

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