Construction of genomic DNA libraries using mucoid vectors
Construction of genomic DNA libraries using mucoid vectors
The steps for constructing genomic DNA libraries in λ phage are essentially the same as in mucosal vectors. In both systems, eukaryotic DNA is ligated to vector DNA in vitro to form a multiplex that can be packaged into λ phage particles. The library constructed in λ phage is stored and proliferated as an infectious recombinant phage. The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition" translated by Huang Peitang et al.
Operation method
Application of mucoid vectors to construct genomic DNA libraries
Principle
The steps for constructing genomic DNA libraries in λ phage are essentially the same as in mucosal vectors. In both systems, eukaryotic DNA is ligated to vector DNA in vitro to form a multiplex that can be packaged into λ phage particles. The library constructed in λ phage is stored and proliferated as an infectious recombinant phage.
Materials and Instruments
T4 Phage DNA ligase Bovine small intestine alkaline phosphatase Bam HⅠ MboⅠ XbaⅠ Restriction endonuclease Move I. Materials For more product details, please visit Aladdin Scientific website.
Chloroform Dephosphorylation buffer Ethanol Phenol Chloroform SM Sodium acetate TE
Agarose gel Pulsed-field gel
1. Buffers and solutions
Chloroform
10 X dephosphorylation buffer (CIP buffer)
Ethanol
Phenol: Chloroform (1:1, V/V)
SM
Sodium acetate (3 mol/L, pH 5.2)
TE ( pH 8.0)
2. Enzyme and buffer
T4 phage DNA ligase
Bovine small intestine alkaline phosphatase (CIP)
Restriction endonucleases: Bam HⅠ, MboⅠ, XbaⅠ
Restriction endonucleases that cut only the vector but not the inserted DNA of the genome.
3. gel
0.7% agarose gel prepared with 0.5 X TBE containing 0.5 μg/ml of EB
0.5% agarose gel prepared with 0.5 X TBE containing 0.5 μg/ml of EB.
Pulsed field gels (or 0.5% agarose gels)
4. culture medium
TB agar plates (with 25 μg/ml kanamycin)
TB medium
TB medium (with 25 μg/ml kanamycin)
5. Nucleic acids and oligonucleotides
Control DNA: λ phage DNA digested with Hind III.
Control DNA: superhelical SupeiCos-1DNA
High molecular mass genomic DNA
Threaded plasmid marker in 1X dephosphorylation buffer
DNA: linear λ phage DNA
6. Specialized equipment
Water baths preset to 16°C and 65°C
7. Vectors and strains
λ Phage Packing Mixture
λ Phage storage solution
E. coli plate strains (e.g. XL1-Blue, ED8767, NM554, DH5αMCR) suitable for determining the titer of the packaged mucoid (e.g. XL1-Blue, ED8767, NM554, DH5αMCR)
SuperCos - 1 DNA (Stratagene) 
II. Methods
Linearization and dephosphorylation of SuperCos - 1 DNA
1. 20 μg of SuperCos-1 DNA was mixed with 50 units of XbaⅠ in 200 μl of 1 X XbaⅠ digestion buffer, and incubated at 37℃ for 2~3 h. The sample was then incubated for 2~3 h at 37℃.
2. After 2 h of incubation, transfer about 1 μl of the reaction solution to another new tube. Analyze the mucoid DNA in this tube by 0.8% agarose gel and use the following controls: (1) 50~100 ng of super-helical SuperCos-1 DNA; (2) 50~100 ng of λ-phage DNA digested by Hind III.
3. Extract the digests once with phenol: chloroform and chloroform, respectively.
4. Transfer the aqueous phase to a new centrifuge tube, precipitate with standard ethanol, wash with 70% ethanol and recover the linear mucoid DNA. open the cap of the centrifuge tube, place it upside down on a piece of paper and allow the ethanol to evaporate, and then dissolve the wet DNA precipitate with 180 μl of H2O. A 100 ng solution of DNA was aspirated and used as a control.
5. Add 20 μl of 10X dephosphorylation buffer to the remaining DNA solution and 0.1 unit of CIP. Incubate the reaction mixture for 30 min at 37 °C. Then incubate the reaction system in a preset 65 °C water bath for 30 min to inactivate the CIP. Two 100 ng portions of the DNA solution were pipetted out and used as controls.
6. The reaction mixture was extracted once with phenol: chloroform and once with chloroform. The linear dephosphorylated SuperCos-1 DNA was recovered by standard ethanol precipitation and washed with 70% ethanol. 180 μl of wet DNA precipitate was dissolved in H2O. 
Isolation of Mucin Arms
7. Transfer 50-100 ng of dephosphorylated DNA solution to a new centrifuge tube and store in an ice bath. Add 20 μl of 10x BamHl restriction buffer and 40 units of BamHⅠ to the remaining dephosphorylated DNA and incubate at 37℃ for 2-3 h.
8. After 2 hours of incubation, transfer 1 portion of DNA solution to another microcentrifuge tube. Analyze the two DNA samples by agarose gel electrophoresis. After digestion with BamH I, the linear 7.9 kb size dephosphorylated SuperCos-1 DNA fragment should be cleaved extensively into two DNA fragments of approximately 1.1 kb and 6.8 kb.
9. Extract the reaction mixture once with phenol: chloroform and once with chloroform. After precipitation with standard ethanol and washing with 70% ethanol, dissolve the moistened DNA precipitate in 20 μl H2O and store at 4 °C.
Partial digestion of high molecular mass genomic DNA
10. Establish the conditions for the partial digestion of a 30 ng sample of high molecular mass genomic DNA with Mbo I. The aim is to produce the maximum number of DNA fragments of 38-52 kb in size.
The course of the digestion reaction can be characterized by pulsed-field gel electrophoresis (PFGE) or 0.5% agarose gel electrophoresis (less effective) with the following markers: (i) unit lengths of λ-phage DNA and/or (ii) λ-phage DNA polymers attached to the cos-site, which are partially digested by a restriction enzyme that cleaves linear λ-DNA only once. 0.5% agarose gel is very thin, so the gel is not suitable for perfusion and electrophoresis. The 0.5% agarose gel is very thin, and gel filling and electrophoresis should preferably be carried out at 4°C, with low voltage (1~2 V/cm) and long time (15~24 h) electrophoresis.
When only a small amount of genomic DNA is available (e.g., when constructing libraries from flow-sorted chromosomes or gel-purified YAC DNA), the conditions for partial digestion can be set up in a small-scale reaction system containing 50~100 ng of genomic DNA and different amounts (0.0005~0.001 units) of restriction enzymes. After 15 min of digestion, a small portion of 10-20 ng of the reaction is analyzed by pulsed-field gel electrophoresis (PFGE) and Southern blotting, where the probes used for Southern blotting have a repetitive sequence with the DNA being detected.
11. According to the partial digestion conditions established in step 10, set up three large-scale reaction systems, each containing 100 μg of high-molecular-quality genomic DNA and the optimal concentration of MboI as determined in step 10. At the end of the incubation period, remove a small amount of each partially digested DNA to identify the size of the molecule by agarose gel electrophoresis, as described in step 10.
12. Combine the two partially digested samples containing the maximum number of genomic DNA fragments of 38 - 52 kb in size. The combined DNA is extracted once with phenol, once with chloroform and once with chloroform. Precipitate with ethanol and wash once with 70% ethanol. Dissolve the moist DNA precipitate with 180 μl TE (pH 8.0).
Dephosphorylation of high molecular mass genomic DNA
13. Add 20 μl of 10 x dephosphorylation buffer and 20 units of CIP to the partially digested genomic DNA resuspended as described above, and quickly draw a small sample of DNA to use as a control.
(1) Pipette a small portion of the reaction containing 0.1 to 1.0 μg of DNA into a small microcentrifuge tube (0.5 ml) pre-filled with 1X dephosphorylation buffer (e.g., Bam HI-cut pUC) containing 0.3 μg of linear plasmid.
(2) Add 0.3 μg of the same linear plasmid to 10 μl of 1x dephosphorylation buffer as another control.
No CIP is added to this control.
(3) Continue with steps 14 to 16.
14. Incubate the mass dephosphorylation reaction and the two control samples at 37°C for 30 min, then transfer to a preset 65°C water bath and incubate for 30 min to inactivate CIP.
15. Cool the reaction to room temperature and extract once with phenol:chloroform and once with chloroform. The reaction was precipitated with ethanol and washed once with 70% ethanol to recover the DNA.
If only a small amount of genomic DNA (<1 μg) is present in the dephosphorylation reaction, dialyze the extracted DNA on a floating filter membrane with TE (pH 8.0).
16. The two controls were lysed in 10 μl of 1X ligation buffer. 0.1 Weiss unit of T4 phage DNA ligase was added to each tube and incubated at room temperature for 3 h. The ligated DNA products were identified by agarose gel electrophoresis.
In control samples containing genomic DNA and plasmid DNA, there should be no evidence of ligation because they have been treated with phosphatase, whereas untreated plasmid DNA should be converted to multimers or closed-loop molecules.
17. Dissolve the DNA in the large-scale reaction system with a small volume of water overnight at 4°C to achieve a final concentration of approximately 500 μg DNA/ml. The DNA concentration can be estimated by agarose gel electrophoresis, preferably to determine A260.
Ligation of Mucin Arms to Genomic DNA: Packaging Reaction and Recombinant Plate Spreading
18. Set up a series of ligation systems (final volume 20 μl), including:
Set up a series of ligation systems (final volume 20 μl), including: 2 μg of fucosylated arm DNA, 0.5, 1.0 or 2.5 μg of dephosphorylated genomic DNA, 2 μl of 10 X ligation buffer, 2 Weiss units of T4 phage DNA ligase, and incubate ligation reagents for 12-16 h at 16℃.
When the two cos sites of the vector used are separated by a flat-end-generating restriction enzyme, the ligation buffer should be supplemented with ATP to a final concentration of 5 mmol/L to inhibit the ligation of flat ends (Feiretti and Sgaramella 1981), which in turn inhibits the formation of the planted multiplex.
19. When using a commercial packaging reaction kit, 5 μl of each ligation reaction is packed into λ phage particles (equivalent to 0.5 μg of vector arm) under the conditions recommended by the supplier. At the end of the packaging reaction, add 500 μl of SM and 20 μl of chloroform to the reaction, and store the dilution at 4°C.
The size of the insert fragment that can be cloned into the mucilage is influenced by the method used to prepare the packaging extract (Bates and Swift 1983). Ideally, extracts for packaging mucilage should be prepared with a buffer containing spermidine rather than putrescine. This system shows selectivity for the size of the DNA molecule being packaged when the humicamine is removed from the packaging extract and the packaging reaction. Thus, when the DNA length is 80% of the wild-type somatic DNA, the packaging efficiency is 1/200 that of the wild-type λ bacteriophage. in the absence of putrescine in the block, the mucilage containing the larger insertion fragment (~45 kb) is preferentially packaged, and for reasons that are not yet known, and possibly due to the lack of ter functionality, some of the packaging extracts that are suitable for the DNA of the λ bacteriophage are not suitable for packaging mucilages. Some commercial packaging extracts, such as Stratagene's Gigapack III XL, are more suitable for mucoid library construction than others.
20. Transduce suitable E. coli host bacteria to determine the titer of the packaging mucilage in each packaging reaction. After a small portion of each reaction was taken and diluted 100-fold, 0.1 ml was mixed with 0.1 ml of SM and 0.1 ml of fresh plate inoculum. The infected culture was incubated at 37°C for 20 min to allow adsorption of phage particles containing recombinant mucoid particles. The kanamycin resistance gene in the SuperCos-1 vector was expressed by adding 1 ml of TB and continued incubation at 37℃ for 45 min.
21. 0.5 ml and 0.1 ml of bacterial cultures were spread on TB agar plates containing 25 μg/ml kanamycin. The plates were incubated at 37°C overnight and then the number of bacterial colonies was counted.
22. 12 single colonies were picked and incubated in TB containing 25 μg/ml of kanamycin in a small scale (2.5 ml) for a period of not more than 6-8 h. The incubation was vigorously shaken.
Isolation and analysis of recombinant mucilage: validity of the library
23. 1.5 ml of each of the above 12 small-scale bacterial cultures was used to isolate mucoid DNA by alkaline lysis.
24. Take 2~4 μl of each prepared DNA and digest it with restriction enzymes (e.g. NotⅠ and SatⅠ) that only cut the fucoidan vector but not the insert fragment of the cloned genomic DNA, and then analyze the size of the digested fragments by 0.7% agarose gel electrophoresis.
25. Calculate the proportion of colonies carrying the insert fragment.
26. Estimate the average length of the insert fragments by isolating some clones and identifying the size of the insert fragments by pulsed-field gel electrophoresis.
27. calculate the "library capacity" of the library, i.e. how many genome equivalents it contains.
If the size and quality of the library are satisfactory, the library can be plated and screened by hybridization, or amplified and stored.
If difficulties are encountered in library construction, try to identify the most likely cause of failure in the experiment. For example, a high proportion of "empty" clones may indicate incomplete dephosphorylation of vector DNA; if there are too few recombinants, this may indicate an insufficient amount of genomic DNA or mucosal arms in the ligation reaction, or inefficient tandem packaging into the λ-phage particle. 