Ligation experiments of λ phage arms to exogenous genomic DNA fragments
When a λ phage arm is attached to an exogenous genomic DNA fragment, two parameters must be taken into account: the molar ratio of the phage arm to the potential insertion fragment, and the concentration of each type of DNA in the reaction mixture. This experiment is based on the "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Operation method
Ligation experiments of λ phage arms to exogenous genomic DNA fragments
Principle
When the λ phage arm is attached to an exogenous genomic DNA fragment, two parameters must be taken into account: the molar ratio of the phage arm to the potential insertion fragment, and the concentration of each type of DNA in the reaction mixture.
Materials and Instruments
Phage T4 DNA ligase Genomic DNA λ Phage packaging mix λ Phage arm DNA Move I. Materials For more product details, please visit Aladdin Scientific website.
ATP SM and SM+ gelatin
Agarose gels LB or NZCYM agar plates Water baths
1. Buffers and solutions
ATP ( 10 mmol/L)
SM and SM+ gelatin
2. Enzyme and buffer
Phage T4 DNA Ligase
3. gels
Agarose gel (0.7%) with 0.5X TBE containing 0.5 μg/ml ethidium bromide.
4. nucleic acids and oligonucleotides
Genomic DNA of appropriate size for vector cloning
5. Culture media
LB or NZCYM agar plates
LB or NZCYM top agarose
6. Specialized equipment
Heater or water bath pre-set at 47°C (for top layer agarose)
Water bath preset at 16°C
7. Carriers and strains
λ Phage Packing Mixture
λ Phage arm DNA
II. Methods
1. Create a series of ligation reactions containing the following:
λ phage arm 0.5~1.0 μg, partially digested genomic DNA 6~1200 ng, 10X ligation buffer 0.5~1.0 μl, 10 mmol/L ATP (if necessary) 0.5~1.0 μl, phage T4 DNA ligase 0.5~1.0 μl, water to 5 or 10 μl.
Set up two control reactions with either vector or insert DNA. Incubate the ligation reaction at 16 ℃ for 4~16 h.
To maximize ligation efficiency, set up the reaction system in the smallest possible volume (5~10 μl). If ATP is added to the reaction mixture as a component of the 10X Ligation Buffer, a larger volume will be reserved for the vector or exogenous DNA.
If a commercial ligation buffer containing ATP is used, omit ATP.
2. Take 10% to 25% of each ligation reaction and package into phage pellets according to the manufacturer's instructions for packaging extracts.
Most manufacturers provide a phage DNA control as a standard for testing packaging efficiency.
3. Perform a 10-fold serial dilution ( 10-1 to 10-5 ) of the packaging reaction, using SM+ gelatin or the appropriate buffer recommended by the manufacturer as the diluent.
4. Analyze the number of phage spots formed at 1 μl and 10 μl of each dilution.
5. From the ligation reaction that yielded the most infectious phage particles, pick 6~12 phage spots and prepare a small amount of recombinant DNA from each.
6. Digestion with appropriate restriction enzymes followed by 0.7% agarose gel electrophoresis and detection of the size of the inserted genomic DNA using appropriate molecular quality standards.
7. If the phage is a recombinant and contains an insert fragment of the appropriate size, a genomic DNA library is constructed by setting up multiple ligation and packaging reactions. The ratio of insert fragments to vector DNA in these reactions should be that used to generate the most recombinant phage spots in the trial reaction.
8. Estimate the amount of all recombinant phage spots generated by large-scale ligation and packaging reactions and calculate the extent to which a library of this size can cover the entire target genome.
In order to provide a 5-fold coverage of a mammalian genome ( 3X109 bp), a λ-phage library containing an average of 20 kb insertion fragments would need to contain 2X106 independent recombinants.