|
Applications
-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
Storage Buffer
20mM Tris-HCl(pH 7.5), 50mM KCl, 1mM DTT, 0.1mM EDTA, and 50% glycerol
Stabilizers
10X Ligation Buffer A
0.4M Tris-HCl, 0.1mM MgCl2, 0.1M DTT, and 5mM 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.
Standard Reaction
2. We recommend using a 1:3 molar ratio of vector to insert DNA when
cloning a fragment into a plasmid vector. These ratios will vary with
other types of vectors.
1. In a microcentrifuge tube prepare a 5-10µl mix in water or TE
buffer of digested vector DNA (50-400 ng) and foreign DNA to be
inserted (a 1:2 or 1:3 molar ratio of vector to insert DNA when
cloning a fragment into a plasmid vector is recommended).
2. Add the following components to the same tube:
i. 2µl 10X Ligase Buffer A
ii. 2µl 10X Ligase Buffer B
iii. 1µl T4 DNA Ligase
iv. Nuclease-Free Water to final volume of 20µl
3. Vortex the tube and spin down in microcentrifuge for 3-5 sec.
4. Incubate the mixture for 5 -20 min at 22°C
5. Inactivate T4 DNA Ligase by heating the reaction mixture at 65°C
for 10 min.
Use the mixture for transformation.
|
