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DNA Ligase 2×In-Fusion Cloning Mix Molecular Biology Reagent

specification:100T
  • G3350-100T
  • Servicebio
  • Servicebio

Product Description

G3350-100T

Introduction:

2×In-Fusion Cloning Mix can quickly ligate and clone single or 2~3 multiple fragments into the vector. It is especially suitable for single fragment ligation, and the efficiency of 2-3 fragment ligation will be reduced. Use any method to linearize the vector,and introduce the end sequence of the linearized vector at the 5'end of the forward/reverse amplification primer of the DNA fragment inserted into the linearized vector, so that the 5'and 3'ends of the PCR amplification product are respectively There is a sequence (15-25 bp) consistent with the end of the linearized vector. In the 2×In-Fusion Cloning Mix premix, mix the PCR product with the same sequence at both ends of the linearized vector and the linearized vector in a certain proportion, and then incubate on ice (or in a mixture of ice and water) for 5 min, you can transform competent cells and complete directional cloning.

This premix can achieve efficient and rapid DNA seamless cloning. And can be used for the construction of recombinant plasmids or gene point mutation recombinant plasmids. The premix does not depend on the ligase system, greatly reducing the background of vector self-ligation, and at the same time, it does not need to consider the restriction endonuclease sites contained in the insert itself. For the construction of single-segment DNA recombinant plasmids, the proportion of positive clones obtained by using this premix is as high as 99%. The use of this premix does not rely on constant temperature equipment, and all operations from DNA sample preparation to transformation and plating of recombinant products can be completed within a few hours, which greatly saves time.

 

Storage and transportation

Transport with wet ice; storage at -20°C, valid for 12 months.

 

Component

G3350-10T

G3350-20T

G3350-100T

2×In-Fusion Cloning Mix

50 μL

100 μL

5×100 μL

pUC19 (Linearized, Control Vector, 5 ng/μL)

-

10 μL

10 μL

Control Insert (10 ng/μL)

-

10 μL

10 μL

User manual

1

 

Reaction system (recommended 10 μL reaction system) 

Component

Volume

2×In-Fusion Cloning Mix

5 μL

Vector

X μL

DNA Segment

Y μL

Nuclease-Free Water

Add To 10 μL

Reaction conditions

Ice-water bath (ice-water mixture), react for 5-10 min.


Reaction product conversion

1. Remove competent cells (such as E.coli DH5α, E.coli Top10, etc.) from the refrigerator at -80°C and thaw them on ice.

2. Add the reacted sample to the competent state, gently move the bottom of the tube with your fingers to mix, and bath in ice for 30 minutes.

3. The product is then placed in a 42°C water bath to heat for 90 seconds. After the completion, it is quickly placed on ice and bathed in ice for 2-5 minutes.

4. Take 900 μL of sterile SOC or LB medium and add it to the EP tube. After mixing, place the EP tube on a shaker and incubate at 220 rpm at 37°C for 1 hour to recover the bacteria (it can also be placed in a 37°C incubator for static Place culture for 1 h);

5. According to experimental requirements, draw different volumes of transformed competent cells and add them to the LB solid medium containing the corresponding antibiotics, spread the cells evenly, and after the liquid is completely absorbed, place the plate upside down in a 37°C incubator and cultivate overnight.

 

Identification of positive clones

The monoclonal colonies grown on the plate are picked for colony PCR identification, or after culture, the plasmid is extracted by restriction digestion or PCR identification, or the extracted plasmid is directly sequenced and analyzed for identification.

 

Precautions

1. The vector and target fragment must be purified by gel and electrophoresis to detect their quality and concentration. When the concentration is low, it is not necessary to add water and directly use it to make up.

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

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

4. When the total volume of the vector and insert is greater than 5 μL, the reaction system can be amplified to 20 μL.

5. The volume of the ligation product should not exceed 1/10 of the volume of the competent cell, otherwise the transformation efficiency will be significantly reduced. The volume of the ligation product and the competent cell can be increased in the same proportion (such as 20 μL of the ligation system to transform 200 μL of competent cells).

6. 2×In-Fusion Cloning Mix is recommended to be taken out when used, and returned to -20°C immediately after use. After thawing, it can be divided into packages and frozen to reduce repeated freezing and thawing times.

7. When electroporation is used for transformation, the reaction product needs to be purified by column method or ethanol precipitation method.

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