T4 DNA Ligase catalyzes the joining of two strands of DNA between the 5'-phosphate and the 3'-hydroxyl groups of adjacent nucleotides in either a cohesive-ended or blunt-ended termini. RBC Rapid DNA Ligation Kit is designed for efficient ligation of DNA inserts into plasmid vectors in just 5 minutes.
- Joining double-stranded DNA with cohesive or blunt termini
- Joining of oligonucleotide linkers to blunt-ended DNA
- Repairing nicks in duplex DNA, RNA or DNA-RNA hybrids
20 mM Tris-HCl (pH 7.5), 50 mM KCl, 1mM DTT, 0.1 mM EDTA, and 50 % glycerol Stabilizers
10X Ligation Buffer A
0.4 M Tris-HCl, 0.1 mM MgCl2, 0.1 M DTT, and 5 mM ATP (pH 5.0 at 250C)The performance of this buffer depends on the integrity of the ATP. Store the buffer in small aliquots at -20░C to minimize degradation of the ATP and DTT.
10X Ligation Buffer B
Contains an enhancer which dramatically increases ligation times for blunt ended DNA.
One unit of enzyme catalyzes the conversion of 1 nanomole of [32PPi] into Norit-adsorbable form in 20 min at 37░C(Weiss unit). We recommend using a 1:3 molar ratio of vector : insert DNA when cloning a fragment into a plasmid vector. These ratios will vary with other types of vectors. In a microcentrifuge tube prepare 5-10ul mix in water or TE buffer of digested vector DNA(50-400ng) and foreign DNA to be inserted. Add the following components to the same tube :
- 10X Ligase Buffer A 2 Ál
- 10X Ligase Buffer B 2 Ál
- T4 DNA Ligase 1 Ál
- Nuclease-Free Water to final volume of 20 Ál
Vortex the tube and spin down in microcentrifuge for 3-5 sec. Incubate the mixture for 5 -20 min at 22░
C. Inactivate T4 DNA Ligase by heating the reaction mixture at 65░
C for 10 minutes Use the mixture for transformation.
|Cat. No. RC0111
Reactions: 100 Ligations
T4 DNA Ligase (3U/ul):100 ul (300 units)
10 X Ligation Buffer A: 200 ul
10 X Ligation Buffer B: 200 ul