λ Alkaline phosphatase treatment assay of phage vector DNA
λ Alkaline phosphatase treatment assay of phage vector DNA
Removal of the terminal 5'-phosphate groups from the inner ends of the two arms of the λphage effectively prevents self-association and reduces the background of non-recombinant phages. This method can be used to suppress the non-recombinant background when using insertion vectors containing a single cloning site (e.g., λ＞10, λ＞11, and λORF8), or when using insertion vectors that contain multiple cloning sites but are cleaved by a single enzyme (e.g., λ＞18-23 and λZAP). This experiment is based on the "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Operation method
Alkaline phosphatase treatment experiments with λ phage vector DNA
Principle
Removal of the terminal 5'-phosphate groups from the inner ends of both arms of the λ phage effectively prevents self-association and reduces the background of non-recombinant phages. This method can be used to suppress the non-recombinant background when using insertion vectors containing a single cloning site (e.g., λgt10, λgt11, and λORF8) or when using insertion vectors that contain multiple cloning sites but are cleaved by a single enzyme (e.g., λgt18-23 and λZAP).
Materials and Instruments
Phage T4 DNA ligase Bovine small intestinal alkaline phosphatase Dephosphorylation buffer Protease K Restriction endonuclease λ Phage DNA λ Phage Packing Mixture Move I. Materials For more product details, please visit Aladdin Scientific website.
λ Compounding buffer Chloroform EDTA Ethanol Phenol: chloroform SDS Sodium acetate TE Tris-Cl
Dog foot nail clippers Water bath
1. Buffers and solutions
(1) λ Compounding buffer
100 mmol/L Tris-Cl ( pH 7.6)
10 mmol/L MgCl2
(2) ATP (10 mmol/L)
(3) Chloroform
(4) EDTA (0.5 mmol/L, pH 8.0)
(5) Ethanol
(6) Phenol: chloroform (1:1, V/V)
(7) SDS (10%, m/V)
(8) Sodium acetate (3 mol/L, pH 5.2 and pH 7.0)
(9) TE (pH 7.6 and pH 8.0)
(10) Tris-Cl (10 mmol/L, pH 8.3)
2. Enzyme and buffer
Phage T4 DNA ligase
Bovine small intestine alkaline phosphatase
10X dephosphorylation buffer (100 mrnol/L Tris-Cl (pH 8.3), 10 mmol/L ZnCl2, 10 mmol/L MgCl2 )
Protease K
restriction endonuclease
3. Nucleic acids and oligonucleotides
λ Phage DNA
4. Specialized equipment
Dog toe-nail clipper (dog toe-nail clipper) or wide-mouth tip
Water bath pre-set at 16°C, 42°C, 56°C, 68°C
5. Vectors and strains
λ Phage packaging mixture
II. METHODS
1. Dissolve 50~60 μg of suitable λ phage vector DNA in λ replication buffer in a final volume of 150 μl. incubate at 42℃ for 1 h to replicate the end of the viral DNA containing the cos site.
2. Add 20 μl of 10X ligase buffer, 20 μl of 10 mmol/L ATP (if necessary), and 0.2-0.5 Weiss units of T4 DNA ligase/μg of DNA, and incubate at 16℃ for 1-2 hours.
3. Extract the ligation reaction with phenol: chloroform.
4. Centrifuge for 1 min at room temperature in a microcentrifuge to separate the organic phase from the hydrophilic phase, and transfer the hydrophilic phase containing viral DNA to a new centrifuge tube. The transfer was accomplished with automated pipetting equipment equipped with disposable tips that had been clipped with dog-leg nail clippers to increase pore size.
5. The DNA was recovered by standard ethanol precipitation, then the precipitate was washed with 1 ml of 70% ethanol and centrifuged for 2 min. The supernatant, 70% ethanol, was removed, and the open centrifuge tube was placed on the bench to allow the ethanol to evaporate, then the DNA precipitate was redissolved in 150 μl of TE (pH 8.0).
6. Digest with one or more restriction enzymes.
7. Repeat steps 3 and 4.
8. Add 0.1 times the volume of 3 mol/L sodium acetate (pH 7.0) and 2 times the volume of ethanol, centrifuge at 4°C for 10 min to recover the DNA, wash with 1 ml of 70% ethanol, centrifuge again for 2 min to remove the ethanol, and place the tube open on a bench to allow the residual ethanol to evaporate.
9. Dissolve the above DNA precipitate in 10 mmol/L Tris-Cl (pH 8.3) to a final concentration of 100 μg/ml. 0.2 μg of the DNA component was taken and stored in an ice bath, and the rest of the DNA precipitate was incubated with an excess of CIF at 37℃ for 1 h as described below:
(1) Add 0.1 times the volume of 10X phosphate removal buffer and 0.01 unit of CIP per 10 μg of λ-phage DNA.
(2) Mix well, incubate at 37℃ for 30 min, add another CIP and continue to incubate for 30 min.
10. Add SDS and EDTA (pH 8.0) to a final concentration of 0.5% and 5 mmol/L, respectively, mix the solution with slight vortexing, add proteinase K to a final concentration of 100 μg/ml, and incubate at 56℃ for 30 min.
11. Cool to room temperature, purify the DNA by extracting once with phenol, once with chloroform and once with chloroform, and recover the DNA by ethanol precipitation in the presence of 0.3 mol/L sodium acetate (pH 7.0).
12. The DNA was dissolved in TE (pH 7.6) at a concentration of 300-500 μg/ml, and this dephosphorylated DNA was subsequently divided into multiple 1-5 μg fractions and stored at -20 °C.
13. The efficiency of dephosphorylation was determined by ligating the digested vector fractions (0.2 μg) before and after CIP treatment. The DNA is packaged into phage particles and the infection titer is determined.
Phosphatase treatment will result in a 2 to 3 order of magnitude decrease in the ligation and packaging efficiency of the phage arms.