Toward Flat-Ended DNA Ligation Synthesis Junction Experiment
Toward Flat-Ended DNA Ligation Synthesis Junction Experiment
A junction is a synthesized DNA fragment that is complementary to itself, usually 8-16 nucleotides in length, and the complementary fragment can be complexed to form a double-stranded molecule with a flat end containing an enzyme cleavage site. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Toward flat-end DNA ligation synthetic junction experiments
Principle
A junction is a synthesized DNA fragment that is complementary to itself, usually 8-16 nucleotides long, and the complementary fragment can be complexed to form a double-stranded molecule with a flat end containing an enzyme cleavage site.
Materials and Instruments
T4 phage ligase T4 phage polyribonucleotide kinase Restriction endonuclease Exogenous or target DNA fragments Move I. Materials For more product details, please visit Aladdin Scientific website.
Amine acetate ATP Ethanol 10X Junction kinase buffer MgCl2 Dithiothreitol (DTT) Bovine serum albumin Phenol Chloroform Sodium acetate TE
10% polyacrylamide gel or 0.7% agarose gel
1. Buffers and solutions
Amyl acetate (4 mol/L, pH 4.8), ATP ( 5 mmol/L and 10 mmol/L) (if ATP is already present in the ligation buffer, omit the addition of 5 mmol/L ATP in step 2), ethanol, 10X Junction Kinase Buffer (600 mmol/L Tris-Cl ( pH 7.6), 100 mmol/L MgCl2. 100 mmol/L dithiothreitol (DTT), 2 mg/ml bovine serum albumin (BSA)), phenol:chloroform (1:1, V/V), sodium acetate (3 mol/L, pH 5.2), TE ( pH 8.0).
2. Enzymes and buffers
T4 phage ligase, T4 phage polyribonucleotide kinase, restriction endonuclease.
3. gels
10% polyacrylamide gel, or 0.7% agarose gel.
4. nucleic acids and oligonucleotides
Exogenous or target DNA fragments (flat ends), synthetic junctions, TE solubilization, concentration 400 μg/ml.
5. Radiolabeling
[ y-32P ] ATP (1~10 μCi).
6. Specialized equipment
Column rotary chromatography device, water bath with adjustable temperature up to 65℃.
II. Methods
1. Phosphorylation of fittings (if required)
(1) Add the following reaction mixture to a sterile 0.5 ml centrifuge tube: 1.0 μl of 10X Junction Kinase Buffer, 1.0 μl of 10 mmol/L ATP, 2.0 μg of synthetic junctions [solubilized in TE (pH 8.0)], make up to 9 μl of H2O, 10 units of T4 phage polyribonucleotide kinase, and 250 pmol of a 12-base junction. The reaction was incubated for 1 h at 37℃.
If necessary, methylation of the internal cleavage site of the target DNA should be carried out at this time. Methylation should be done according to the seller's instructions before ligating the junctions.
2. Phosphorylation of the junction to the flat end of the DNA
(1) Calculate the concentration of DNA flat ends in the preparation, then add the following ligation reaction mixture to a 0.5 ml centrifuge tube: 50 μg of 1 kb long double-stranded DNA fragments = 78.7 nmol or 157.4 nmol/L ends; 2 pmol ends of flat-end DNA; 150-200 pmol ends of phosphorylated junctions; make up 7.5 μl of H2O; 10X Ligase Buffer 1 μl of phosphorylated junctions. 10X Ligase Buffer 1.0 μl; 5 mmol/L ATP 1.0 μl; T4 Phage DNA Ligase 1.0 Weiss unit.
The reaction mixture was incubated at 4 ℃ for 12~16 h. The reaction mixture was then incubated for 12~16 h at 4 ℃.
(2) The reaction mixture was incubated at 65℃ for 15 min to inactivate T4 phage DNA ligase.
(3) Allow the ligation mixture to cool to room temperature, then add 10 μl of 10x endonuclease buffer, 50 units of restriction endonuclease and 100 μl of sterilized water.
Allow the reaction to incubate at 37℃ for 1~3 hours.
3. Recovery of ligation products (DNA)
(1) Purify the digested DNA fragments by extraction with phenol and chloroform, then add amyl acetate (final concentration 2 mol/L) and 2 times the volume of ethanol to precipitate the DNA.
(2) Collect the DNA precipitate by centrifugation at high speed for 15 min at 4 ℃, and add 50 μl TE (pH 8.0) to dissolve the precipitate.
(3) The DNA solution was passed through a centrifugal chromatography column to remove the excess junctions.
(4) The DNA was recovered by ethanol precipitation, and the precipitate was re-dissolved with 10-20 μl TE (pH 8.0).
The modified DNA fragments can now be ligated into plasmid (or phage) vectors (their protruding ends are complementary to the sequence introduced by the junctions in the DNA fragments).