Replacement vectors (e.g., λ2001, λDASH, EMBL series, Charon 34, Charon 35, and Charon 40) contain a series of restriction sites that are aligned in opposite directions at the ends of the central filler fragment (Frischaufet al. 1983). The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition" Translated by Huang Peitang et al.

Operation method

Preparation of λ phage DNA by double restriction enzyme cleavage for use as a cloning vector in the experiment

Principle

Displacement vectors (e.g., λ2001, λDASH, EMBL series, Charon 34, Charon 35, and Charon 40 ) contain a series of restriction sites that are arranged in opposite orientations at the ends of the central filler fragment (Frischaufet al. 1983).

Materials and Instruments

Phage T4 DNA ligase Restriction endonuclease λ Phage Packing Mixture
ATP Chloroform Ethanol Phenol: Chloroform Sodium acetate Caramel Gel Sampling Buffer
Agarose Gel

Move

I. Materials

1. Buffers and solutions

ATP ( 10 mmol/L)

Chloroform

Ethanol

Phenol: Chloroform (1:1, V/V)

Sodium acetate (3 mol/L, pH 5.2)

Sucrose Gel Sampling Buffer

TE ( pH 7.6 and pH 8.0)

2. enzymes and buffers

Phage T4 DNA Ligase

restriction endonuclease

3. gels

Agarose gel (0.7%) with 0.5X TBE containing 0.5 μg/ml ethidium bromide

4. nucleic acids and oligonucleotides

λ Phage (replacement vector) 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 purified from the displacement vector is mixed with TE ( pH 8.0 ) to a final volume of 170 μl.

2. Add 20 μl of either 10X restriction enzyme buffer. Take two portions of undigested λ-phage DNA, each containing 0.2 μg, and store in an ice bath.

3. Add a 4-fold excess (100~200 units) of either of the appropriate restriction enzymes and digest for 4 h at the temperature recommended by the manufacturer.

4. Cool the reaction to 0°C in an ice bath. Remove the two 0.2 μg fractions and incubate one of them (save the other for analysis in Step 10) with the other undigested DNA (Step 2) at 68°C for 10 min to break the sticky ends of the phage DNA. Add a small amount (10 μl) of Sucrose Gel Sampling Buffer and immediately add the sample to a 0.7% agarose gel.

5. Purify the DNA by two extractions with phenol: chloroform and one extraction with chloroform.

6. Recover the DNA by standard ethanol precipitation.



7. Re-dissolve the DNA in TE (pH 8.0) at a concentration of 250 μg/ml, add the appropriate 10X restriction enzyme buffer and digest with a second restriction enzyme, again with a 4-fold excess of the enzyme, for 4 h.

8. The DNA was purified by two extractions with phenol: chloroform and one extraction with chloroform, and recovered by standard ethanol precipitation.

9. Re-dissolve the DNA in TE (pH 7.6) at a concentration of 300-500 μg/ml, and store one fraction (0.2 μg) at -20°C.

10. To test the effectiveness of the digestion process, perform a pre-ligation reaction with 0.2 μg of vector digested only by the first enzyme (the fraction set aside in step 4) and 0.2 μg of vector made at the end (step 9 ). An equal amount (0.1 μg) of DNA was taken from each ligation mixture for packaging, and the titer of the phage particles obtained was determined.

(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 10 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.

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