DNA Ligation

DNA ligases allow for the enzymatic sealing of nicks between adjacent residues of a single-strand break on a double-strand substrate and the joining of double-strand breaks.

The establishment of a phosphodiester bond between the 3' hydroxyl and 5' phosphate of adjacent DNA residues occurs in three steps:

  • Firstly, the ligase is self-adenylated following a reaction with free ATP.
  • Next, the adenyl group is transferred to the 5'-phosphorylated end of the "donor" strand.
  • Finally, the formation of the phosphodiester bond proceeds after the reaction of the adenylated donor end with the adjacent 3' hydroxyl acceptor and the release of AMP.

    In living organisms, DNA ligases are essential enzymes with roles in DNA replication and repair. The Assay Genie Rapid DNA Ligation Kit enables a quick sticky-end or blunt-end DNA ligation in only 5 minutes at room temperature.

Assay Genie Rapid DNA Ligation Kits

Product Code Product Name

MORV0109


GenieClone 5 minute Ligation Mix

GenieClone 5 minute Ligation Mix is a ready-to-use 2 × premix solution, containing T4 DNA ligase that catalyzes the formation of a phosphodiester bond between adjacent 5'-phosphate and 3'-hydroxyl termini in double-strand DNA or RNA. This kit is suitable for sticky end ligation, blunt end DNA ligation and TA ligation. Buffer with optimized ligation enhancer make the reaction more efficient and convenient. Ligation can be completed in 5 min at 25℃. The product can be used directly to transform many chemically competent cells.

GenieClone 5 minute Ligation Mix - Applications

  • Sticky end ligation
  • Blunt end ligation
  • TA ligation Linker
  • Adapter ligation

Quick Ligation Kit - Features & Benefits

  • Quick: ready-to-use and takes only 5 minutes.
  • Convenient: ligation can be performed at 25℃.
  • Flexible: kit can be used for all common ligation reactions.
  • Easy: kit contains all buffers and enzymes required.

Rapid DNA Ligation Kit Protocol

Summary of the Experimental Process:

Figure 1: Process summary of 5min Universal Ligation Mix


1. Ligation Reaction

Component Amount

1.

Prepare the rection mix on ice:

Component Volume

Vector

0.03 pmol

Fragment

0.03 – 0.3 pmol

2 x Universal Ligation Mix

5 µl

ddH2O

To 10 µl

Note:

The optimal vector usage (0.03 pmol) = [0.02 × base pair of vector] ng
The optimal fragment usage (0.09 pmol) = [0.06 × base pair of fragment] ng

2.

Mix thoroughly by pipetting up and down several times (do not vortex), and centrifuge briefly to collect the reaction liquid.

3.

Incubate 5 min at 25℃; Cool down to 4℃ or place on ice immediately.

Other conditions for ligation: overnight at 4℃ or 16℃.

When the total volume of vector and fragment is more than 5 μl, the reaction system can be enlarged to 20 μl. For blunt end ligation or TA ligation, the reaction time can be prolonged to 2 h to improve the efficiency.

The product need to be purified by spin column or chloroform extraction before electrotransformation.

The product can be stored for one week at -20℃.


2. Transformation Protocol

Component Amount

1.

Chemically competent cells for cloning are thawed on ice

2.

Add 5 - 10 μl of the ligation product into 100 μl of competent cells, and mix by fingerflicking (do not vortex the tube), then place the tube on ice for 30 min.

  • The transformed volume of the ligation product should not exceed 1/10 of the volume of competent cells.

3.

Heat shock at 42℃ for 30 sec, then return the tube to ice for 2 - 3 min immediately.

Add 900 μl of SOC or LB liquid medium (without antibiotics), and incubate for 1 h at 37℃ with rotation or shaking (200 - 250 rpm).

Warm selection plates at 37℃.

Centrifuge at 5,000 rpm for 5 min and discard 900 μl supernatant. Resuspend the bacteria and coat onto plates

Incubate overnight at 37℃.


3. Identification of positive clones

PCR identification; Digestion identification; Plasmid identification; DNA Sequencing.