Southern Blot Protocol
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SOUTHERN BLOT PROTOCOL
Following electrophoresis and staining of DNA, cut the agarose gel to size by removing the wells at the top of the gel (to prevent buffer transfer through the wells), and any blank or unrelated lanes that do not need to be transferred.
UV treat for 5 minutes on the transilluminator. (Optional alternative for cellular, i.e. not cloned, DNA: soak the gel in 0.2M HCl for 7 minutes).
Soak the gel in base solution for 45 minutes, followed by the neutralizing solution for 90 minutes (or 2x the length of time of the base treatment). The gel may be stored in the neutralizing solution for many hours.
Cut two Whatman 3MM filters the same size as the gel, and another to act as a wick, running under the gel and extending into troughs containing the transfer solution. Cut a nitrocellulose or nylon membrane using the 3MM filters as a template to the size of the gel. Nylon membranes are preferred due to their strength upon repeated handling. Handle nitrocellulose and nylon membranes by their edges with tweezers.
Pre-wet the wick in 20x SSPE and position its ends in a reservoir that contains about 500 ml of 20x SSPE. Turn the gel upside down on the wick so that the smooth side will face the nitrocellulose or nylon membrane. Avoid trapping between the gel, membrane and filter paper.
Pre-wet the nitrocellulose in 5x SSPE, or the nylon membrane in distilled water. Place the membrane on top of the gel so that its edge is aligned with the cut wells. Cover this membrane with the two Whatman 3MM filters that were pre-wet in 20x SSPE. Place a few inches of paper towels and a weight (about 1 kg) on top of this. Make sure the paper towels do not hang into the reservoir or lay on the wick. Serological plastic pipets or Saran wrap are useful to avoid this problem
Allow genomic DNA to transfer overnight, or cloned DNA to transfer at least 4 hours (overnight is fine).
Before removing the membrane after the transfer, mark the position of the wells with a pencil. Also initial and date the membrane. Soak the membrane in 5x SSPE for 30 minutes. Blot with a Whatman 3MM filter paper to remove excess moisture.
For nylon membranes only, place the membrane in Saran wrap, and UV treat for 2 to 5 minutes with the DNA side facing the UV light. For nitrocellulose membranes only, bake in an 80°C vacuum oven a few hours until the filters are dry.
Place the membrane in Saran wrap and store at 4°C.
Hybridize the membranes with a labeled probe.
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RECIPES
Treatment Solution (1.5M NaCl, 0.5M NaOH) for Southern and Benton Davis blots.
For 2 liters:
175.35 g NaCl
40 g NaOH
QS to 2 liters with water and autoclave.
Neutralizing Solution
5 M Tris-HCl, 3M NaCl, pH 7.4) for Southern and Benton Davis blots.
For 2 Liters:
121.1 g Tris
350.7 g NaCl
Adjust pH to 7.4 with concentrated HCl (c. 70 ml?) and QS to 2 liters with water.
20X SSPE (3 M NaCl, 0.2 M NaH2PO4, 20 mM EDTA, pH 7.0)
350.7 g NaCl
55.2 g NaH2PO4(H20) (--or-- 48 g anhydrous NaH2PO4, mw = 120.0)
14.89 g EDTA
Dissolve in boiling water, cool to room temperature, & adjust pH to 7.0 with NaOH. QS to 2 liters with water and autoclave.
Following electrophoresis and staining of DNA, cut the agarose gel to size by removing the wells at the top of the gel (to prevent buffer transfer through the wells), and any blank or unrelated lanes that do not need to be transferred.
UV treat for 5 minutes on the transilluminator. (Optional alternative for cellular, i.e. not cloned, DNA: soak the gel in 0.2M HCl for 7 minutes).
Soak the gel in base solution for 45 minutes, followed by the neutralizing solution for 90 minutes (or 2x the length of time of the base treatment). The gel may be stored in the neutralizing solution for many hours.
Cut two Whatman 3MM filters the same size as the gel, and another to act as a wick, running under the gel and extending into troughs containing the transfer solution. Cut a nitrocellulose or nylon membrane using the 3MM filters as a template to the size of the gel. Nylon membranes are preferred due to their strength upon repeated handling. Handle nitrocellulose and nylon membranes by their edges with tweezers.
Pre-wet the wick in 20x SSPE and position its ends in a reservoir that contains about 500 ml of 20x SSPE. Turn the gel upside down on the wick so that the smooth side will face the nitrocellulose or nylon membrane. Avoid trapping between the gel, membrane and filter paper.
Pre-wet the nitrocellulose in 5x SSPE, or the nylon membrane in distilled water. Place the membrane on top of the gel so that its edge is aligned with the cut wells. Cover this membrane with the two Whatman 3MM filters that were pre-wet in 20x SSPE. Place a few inches of paper towels and a weight (about 1 kg) on top of this. Make sure the paper towels do not hang into the reservoir or lay on the wick. Serological plastic pipets or Saran wrap are useful to avoid this problem
Allow genomic DNA to transfer overnight, or cloned DNA to transfer at least 4 hours (overnight is fine).
Before removing the membrane after the transfer, mark the position of the wells with a pencil. Also initial and date the membrane. Soak the membrane in 5x SSPE for 30 minutes. Blot with a Whatman 3MM filter paper to remove excess moisture.
For nylon membranes only, place the membrane in Saran wrap, and UV treat for 2 to 5 minutes with the DNA side facing the UV light. For nitrocellulose membranes only, bake in an 80°C vacuum oven a few hours until the filters are dry.
Place the membrane in Saran wrap and store at 4°C.
Hybridize the membranes with a labeled probe.
--------------------------------------------------------------------------------
RECIPES
Treatment Solution (1.5M NaCl, 0.5M NaOH) for Southern and Benton Davis blots.
For 2 liters:
175.35 g NaCl
40 g NaOH
QS to 2 liters with water and autoclave.
Neutralizing Solution
5 M Tris-HCl, 3M NaCl, pH 7.4) for Southern and Benton Davis blots.
For 2 Liters:
121.1 g Tris
350.7 g NaCl
Adjust pH to 7.4 with concentrated HCl (c. 70 ml?) and QS to 2 liters with water.
20X SSPE (3 M NaCl, 0.2 M NaH2PO4, 20 mM EDTA, pH 7.0)
350.7 g NaCl
55.2 g NaH2PO4(H20) (--or-- 48 g anhydrous NaH2PO4, mw = 120.0)
14.89 g EDTA
Dissolve in boiling water, cool to room temperature, & adjust pH to 7.0 with NaOH. QS to 2 liters with water and autoclave.
