Using a Nitrocellulose Filter

Southern blotting is the term given to the procedure of transferring DNA from a gel matrix to a solid inert filter. DNA samples, fragmented by restriction digests or PCR amplified, are separated by agarose gel electrophoresis (0.5-2.5% agarose), stained with ethidium bromide (0.5 pg/mL) and photographed to record the position of molecular weight markers and the fragments of interest. The gel is rinsed, the DNA depurinated by a HCl (0.25 M) wash, and denatured by washing twice in a denaturing buffer (1.5 M NaCl, 0.5 M NaOH). For the DNA to be transferred to the nitrocellulose filter, it is necessary to neutralize it by washing twice in a neutral, high-salt buffer (1.5 M NaCl; 0.5 M Tris-HCl, pH 7.0). For all the washes, use 5- to 10-fold gel volumes of each buffer and incubate at room temperature for 20-30 min with gentle shaking.

To transfer the DNA to the filter, assemble the blot apparatus as shown in Fig. 1. Fill a tray/sandwich box with transfer buffer (20x SSC; 3 M NaCl, 0.3 M trisodium citrate) and place a 20x SSC-soaked wick (three sheets of Whatman 3MM paper) onto the glass support so that each end dips into the 20x SSC reservoir. The nitrocellulose filter should be cut to the exact size of the gel and wetted by floating on distilled water prior to soaking in 20x SSC. Expel any air bubbles trapped beneath the wick with a gloved hand before placing the gel on to the saturated wick. Apply the 20x SSC-soaked filter to the top of the gel and remove any bubbles as instructed earlier. To prevent the filter from touching the wick, use saran wrap to form a barrier around the edges of the gel. Place five sheets of dry 3MM paper onto the filter and stack paper towels (4-5 cm) on top, cover with a glass plate, and place

Weight <0.75g

Saran wrap-te.

Wick

Wick

Fig. 1 Overview of nucleic acid blotting procedure.

, Tray filled with transfer buffer

Glass plate

Fig. 1 Overview of nucleic acid blotting procedure.

a small weight (up to 500 g) at the top. The apparatus should be left overnight for optimal DNA transfer.

Disassemble the blotting apparatus, leaving a compacted gel/filter amalgamation. It is paramount to mark the position of the wells on the filter and to nick one of the corners to allow correct orientation. To remove excess salt and prevent the filter from becoming brittle, peel the filter from the gel and soak in a 6 x SSC solution for 3 min before drying it on 3MM paper. It is recommended to confirm the DNA transfer by restaining the gel with ethidium bromide. After the filter has dried, sandwich it between two sheets of 3MM paper and bake it for 2-3 hr at 80°C under a vacuum to immobilize the DNA by expelling any water. The filter is now ready for probe hybridization, although it can still be stored for several months in a dry, dark location.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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