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DNA Ligase 2 x Universal Ligation Mix with T4 DNA Ligase and buffer molecular biology reagent

specification:100T
  • G3341-100
  • Servicebio
  • Servicebio

Product Description

G3341-100

Description:

 

2×Universal Ligation Mix is a ready-to-use 2×premix containing T4 DNA Ligase and reaction buffer. The contained T4 DNA Ligase can catalyze the 5'-P and 3'ends of sticky ends or blunt-ended double-stranded DNA or RNA. The -OH ends are combined with phosphodiester bonds. and can repair single-stranded nicks in double-stranded DNA, RNA, and DNA/RNA hybrid chains. The optimized premixed reaction buffer makes the reaction more efficient and convenient to handle. The reaction system can transform a variety of chemically competent cells after reacting at 25°C for 5 minutes.

 

Storage and transportation

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

 

Component

G3341-50

G3341-100

2×Universal Ligation Mix

250 μL

2×250 μL

Connection system (recommended 10 μL reaction system)

Component

Volume

2×Universal Ligation Mix

5 μL

Vector

X μL

DNA segment

Y μL

Nuclease-Free Water

Add To 10 μL

Ligation reaction conditions

The ligation reaction of the sticky end at 25°C is 5-30 minutes, and the ligation reaction of the blunt end at 25°C does not exceed 2 hours or the reaction at 4°C overnight.

Conversion of ligation products

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 well, and ice bath for 30 minutes;

3. The product is then placed in a 42°C water bath for 90 s to heat shock, and after that, it is quickly placed on ice for 2-5 min;

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. It is recommended to configure the reaction system on ice.

2. The vector and target fragments must be gel purified and electrophoresis to detect their quality and concentration. When the concentration is low, you can directly use them to make up without adding water. When the total volume of the vector and insert is greater than 5 μL, the reaction system can be amplified to 20 μL.

3. It is recommended that the molar ratio of vector to insert is 1:3~1:10.

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

5. 2×Universal Ligation Mix is recommended to be taken out when used, and immediately returned to -20°C after use. After thawing, it can be packed and frozen to reduce the number of repeated freeze-thaw cycles.

6. When connecting a blunt-ended vector to a DNA fragment, the vector must be dephosphorylated (G3400 recommended) to prevent its self-circulation.

7. Please wear laboratory clothes and disposable gloves during operation.

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