Preparation of λ phage DNA that can be cleaved by a single restriction enzyme and used as a cloning vector
In some cases, prepared λ phage DNA can be used for cloning after simple digestion with restriction enzymes. However, this option is only feasible if the vector used can be genetically screened for recombinant phages containing exogenous DNA sequences (e.g., EMBL series, λ2001, λDASH, λZAP, λgt10). Because in this case, it is not necessary to take steps to reduce the formation of non-recombinant phage. The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition" translated by Huang Peitang et al.
Operation method
Experiments on the preparation of λ phage DNA that can be cleaved by a single restriction enzyme for use as a cloning vector
Principle
In some cases, prepared λ phage DNA can be used for cloning after simple digestion with restriction enzymes. However, this option is only feasible if the vector used can be genetically screened for recombinant phages containing exogenous DNA sequences (e.g., EMBL series, λ2001, λDASH, λZAP, λgt10). This is because in this case, no steps need to be taken to reduce the formation of non-recombinant phages.
Materials and Instruments
Phage T4 DNA ligase Restriction endonuclease with appropriate buffer Move I. Materials For more product details, please visit Aladdin Scientific website.
ATP Chloroform EDTA Ethanol Phenol: Chloroform Sodium Acetate Caramel Gel Sampling Buffer
Agarose Gel
1. Buffers and solutions
ATP ( 10 mmol/L )
Chloroform
EDTA ( 0.5 mol/L, pH 8.0)
Ethanol
Phenol: chloroform (1:1, V/V)
Sodium acetate (3 mol/L, pH 7.0) (It is important to use sodium acetate at pH 7.0 rather than the commonly used pH 5.2. This is because for EDTA, which is used to remove divalent cations from the buffer after digestion, it will precipitate out of solution at pH 5.2 and at concentrations greater than 5-10 mmol/L).
Caramel gel loading buffer (sucrose gel-loading buffer)
TE (pH 7.6 and pH 8.0)
2. enzymes and buffers
Phage T4 DNA Ligase
Restriction endonucleases with appropriate buffers
3. gels
Agarose gel (0.7%) with 0.5X TBE containing 0.5 μg/ml ethidium bromide
4. nucleic acids and oligonucleotides
λ Phage DNA
5. Specialized equipment
Water bath pre-set at 68°C
6. Carriers and strains
λ Phage Packing Mixture
II. METHODS
1. 25-50 μg of λ phage DNA was mixed with TE (pH 8.0) to a final volume of 170 μl.
2. Add 20 μl of 10X restriction enzyme buffer. Take two portions of undigested phage DNA, each containing 0.2 μg, and store in an ice bath.
3. Add 3 times excess (75~150 units) of suitable restriction enzyme and digest for 1 h at the temperature recommended by the manufacturer.
4. Cool the reaction to 0°C in an ice bath. Remove a 0.2 μg portion and incubate it with another portion of undigested DNA (Step 2) at 68℃ for 10 min to break the sticky ends of phage DNA. Add a small amount (10 μl) of Sucrose Gel Sampling Buffer and immediately add the sample to a 0.7% agarose gel.
If the restriction enzyme digestion is complete, no DNA will migrate to the undigested control band. Instead, two or more (depending on the number of cleavage sites in the vector) smaller DNA fragments will be seen. Careful examination of the number of these small fragments confirms the absence of a partial cleavage product.
This step is not as simple as it sounds. λ The left and right arms of the phage DNA have complementary 12-base long ends that can recombine with each other to form hydrogen bonds, and the resulting DNA can easily be mixed with the uncut phage DNA. For this reason, the DNA samples should be sampled and subjected to agarose electrophoresis as soon as they are removed from the 68℃ water bath.
If digestion is incomplete, heat the reaction to the appropriate temperature, add restriction enzymes (50-100 units), and continue incubation at the optimal temperature recommended by the manufacturer.
5. When digestion is complete, add 0.5 mol/L EDTA ( pH 8.0) to a final concentration of 5 mmol/L, and extract the digested mixture once each with phenol: chloroform and chloroform.
6. Recover the DNA from the hydrophilic phase by ethanol precipitation in the presence of 0.3 mol/L sodium acetate (pH 7.0). The precipitate was collected by centrifugation in a microcentrifuge at maximum speed for 2 min at 4°C. After washing with 70% ethanol, the DNA was redissolved in 100 μl of TE (pH 7.6) and the concentration was determined by measuring the absorbance at 260 nm.
7. A 0.5 μg portion of DNA was taken and tested for ligation capacity as described below:
(1) Adjust the volume of DNA solution to 17 μl with water.
(2) Add 2 μl of 10X Ligation Buffer and, if necessary, 1 μl of 10 mmol/L ATP.
Some commercial ligation buffers contain ATP; if such a buffer is used, it is not necessary to add additional ATP.
(3) Take 5 μl of the mixture made in step (2) and store in an ice bath.
(4) To the remaining mixture (step (2)), add 0.2-0.5 weiss units of T4 DNA Ligase and incubate for 2 h at 16°C.
(5) Package 0.1 μg of ligated and unligated samples and 0.1 μg of undigested vector DNA with a commercial λ-phage packaging reaction.