PFGE is used to isolate very large DNA molecules and thus requires standards of very high molecular mass. Such standards can be obtained from phages such as T7 (40kb), T2 (166kb), and G (758kb) following the method described by Lauer et al. (1977). But a better standard for series with wider coverage is the series of tandems of λ phage DNA. The specific steps described later are a modification of the Vdlrath and Davis (1987) method. This experiment is based on the "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.

Operation method

Molecular quality criteria for pulsed-field gel electrophoresis

Principle

PFGE is used to isolate very large DNA molecules and thus requires standards of very high molecular mass. Such standards can be obtained from phages such as T7 (40kb), T2 (166kb), and G (758kb) following the method described by Lauer et al. (1977). But a better standard for series with wider coverage is the series of tandems of λ phage DNA. The specific steps described later are a modification of the Vdlrath and Davis (1987) method.

Materials and Instruments

Phage T4 DNA ligase Purified phage λDNA
ATP Dithiothreitol EDTA Ligation Buffer Low Melting Point Agarose Buffer Polyethylene Glycol TE
Low Melting Point (LMT ) Agarose Tygon Tubes Water Baths

Move

I. Materials

1. Buffers and solutions

ATP ( 0.1 mol/L)

Dithiothreitol ( 1 mol/L)

EDTA ( 0.5 mol/L, pH 8.0)

1X ligation buffer with polyethylene glycol (50 mmol/L Tris-Cl (pH 7.6), 1 mmol/L ATP, 10 mmol/L dithiothreitol (DTT), 10 mmol/L MgCl₂, 2% PEG 8000)

Low melting point agarose buffer (100 mmol/L Tris-Cl (pH 7.6), 20 mmol/L MgCl₂ )

MgCl₂ ( 20 mmol/L)

Polyethylene glycol (8%, m/V, PEG 8000 solution)

TE (pH 7.6)

2. Enzyme and buffer

Phage T4 DNA Ligase

3. gels

Low melting point (LMT ) agarose

4. Nucleic acids and oligonucleotides

Purified phage λDNA

5. Specialized equipment

Tygon tubes

56°C water bath

II.

1. Dissolve 0.1 g of low melting point agarose in 10 ml of LMT buffer by heating in a boiling water bath or microwave. Cool to 37°C.

2. Dissolve 10 μg of purified phage λDNA in 172.5 μl of TE (pH 7.6), and heat the solution to 56 °C for 5 min.

3. Cool the solution to 37°C and add the following reagents in rapid sequence:

8%PEG8000 62.5 μl

20 mmol/L MgCl₂ 5.0 μl

0.1 mol/L ATP 5.0 μl

1 mol/L DTT 5.0 μl

Phage T4 DNA Ligase 0.5 Weiss unit

1% LMT agarose solution 250 μl

Polyethylene glycol acts as a pooling agent to increase ligation efficiency.

4. The mixture is pipetted into a slightly shorter Tygon tube and cooled on ice until the agarose has completely condensed.

5. Transfer the gel plug into a sterile, disposable plastic tube containing 3x the volume of 1x Ligation Buffer with polyethylene glycol.

6. Allow the gel plugs to incubate in the ligation buffer for 24 h at room temperature, then transfer to a 10-fold volume of 20 mmol/L EDTA (pH 8.0) in a test tube.

7. incubate in EDTA for 1 h, then transfer the gel plugs to a fresh 10x volume of 20 mmol/L EDTA (pH 8.0).

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