Southern blotting (DNA transfer from one agarose gel to two membranes simultaneously)

Summary

DNA can be transferred from both sides of the agarose to both membranes simultaneously. This method is useful when the same restriction endonuclease fragment needs to be analyzed with two different probes.The DNA fragments are transferred at a faster rate, but less efficiently because the liquid in the gel flows out from both sides at the same time, resulting in too rapid a dehydration rate. This method is most efficient when the concentration of the target sequence is high. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Peitang Huang et al.

Operation method

Southern blotting (DNA transfer from one agarose gel to two membranes simultaneously)

Principle

DNA can be transferred from both sides of the agarose to both membranes simultaneously. This method is useful when the same restriction endonuclease fragment needs to be analyzed with two different probes; the DNA fragments are transferred at a faster rate, but less efficiently because the liquid in the gel flows out of both sides at the same time, resulting in too fast a dehydration rate. This method is most efficient when the concentration of the target sequence is high, such as when analyzing cloned DNA (plasmid, phage, mucilage, PAC or BAC) or less complex genomes. Mammalian genomic DNA transferred by this method is so small that single-copy sequence hybridization signals cannot be duplicated or detected in time.

Materials and Instruments

Restriction endonuclease DNA molecular marker Target DNA
Denaturing solution HCl Neutralization buffer Neutral transfer buffer SSC Sucrose gel spike buffer SYBR Gold or ethidium bromide
Agarose gel without ethidium bromide Cross-linking equipment Glass baking dish Nylon or nitrocellulose membrane Rotary oscillation platform Thick absorbent paper Fluorescently labeled transparent ruler

Move

I. Materials

1. Buffers and solutions

Denaturing solution (1.5 mol/L NaCl, 0.5 mol/L NaOH)

HCl ( 0.2 mol/L) applied to DNA depurination

Neutralization buffer (1 mol/L Tris ( pH 7.4), 1.5 mol/L NaCl)

Neutralization buffer (10X SSC)

6X SSC

6X Sucrose Gel Sampling Buffer

SYBR Gold or Ethidium Bromide

2. Enzyme and buffer

Appropriate restriction endonucleases

3. gels

0.7% agarose gel prepared with 0.5X TBE or 1X TAE without ethidium bromide.

4. nucleic acids and oligonucleotides

DNA molecular markers

Target DNA

5. Specialized equipment

Cross-linking equipment (e.g. Stratalinker, Stratagene; GS Gene Linker, Bio-Rad), or microwave oven, or vacuum oven

Glass baking dish

Glass plates

Nylon or nitrocellulose film

Rotary oscillation platform

Thick absorbent paper (e.g., Whatman 3 MM, Schleicher&Schuell GB004, or Sigma QuickDraw)

Transparent ruler with fluorescent markers

Heavy weight (400 g )

II. METHODS

1. Digest the DNA and separate the DNA fragments by gel electrophoresis as described in steps 1-3 of the protocol "Southern blot (capillary transfer of DNA to membrane)".

2. After separation of DNA by gel electrophoresis, stain with ethidium bromide or SYBR Gold and take a photograph. A transparent fluorescent ruler is placed on the edge of the gel so that the size of the DNA bands can be read directly from the photographs based on the migration distance. Prepare the gel for transfer under neutral conditions.

3. Using a clean scalpel or paper cutter, cut two nylon or nitrocellulose membranes 1 to 2 mm larger than the gel on each side. Cut one corner of the membrane to correspond to the cut corner of the gel. Cut 4 sheets of thick blotting paper the same size as the membrane.

4. Float the membrane in a dish of deionized water until it is completely wet from the bottom up, then immerse the membrane in 10 X SSC for at least 5 min.

5. Pipette each layer to dislodge air bubbles, especially between the membrane and the gel. Place one membrane on two sheets of moist blotting paper. Place the gel on top of the membrane with the cut off corner of both aligned. Immediately place another membrane on the other side of the gel, followed by two sheets of moist blotting paper.

6. Place the blotting paper, membrane, and gel onto a 5-10 cm high stack of paper towels. Place another stack of paper towels on top. Place a glass plate on top of the paper towels and compact it with a 400 g weight.

7. After 2-4 h, remove the blotting paper and paper towels. Transfer the gel and membrane to dry blotting paper and mark the approximate location of the gel spiking holes with a very soft pencil or ballpoint pen.

8. Fix the DNA on the membrane.

9. Hybridize the probe directly to the immobilized DNA.

Any membrane not immediately used for hybridization should be well dried, loosely wrapped in aluminum foil or blotting paper, and preferably stored under vacuum at room temperature.

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