DNA Amplification Fingerprinting Protocol
The PCR reaction mix (10 µL) contains template DNA (2 ng/µL), primer (0.3 µM), Taq DNA polymerase, Stoffel Fragment (Perkin Elmer; 5 U), Mg++ (2.5 mM), buffer (1X) and overlaid with a drop of mineral oil. Amplifications are performed using the 96-well plates in a MJ Research thermal cycler for 35 cycles after an initial denaturation at 94C for 5 min and a final extension at 72C5 min. For arbitrary and mini-hairpin primers, each cycle consists of 5 sec at 94C, 20 sec at either 35 C or 45C (depending on the primer; see Table 2) and 30 sec at 72C For SSR primers, each cycle is 1 min at 94C, 1 min at 55C and 2 min at 72C.
DNA fragments are separated in a vertical electrophoresis system using a polyacrylamide-based vinyl polymer (GeneAmp; Perkin Elmer, Norwalk, CT); (He et al., 1994). Gels were prepared as follows:
- Add 3.5 mL deionized water to a clean beaker (10 mL).
- Add 1.25 mL of GeneAmp Detection Gel solution and 0.25 mL of 10X TBE buffer (1M Tris. HCl, 0.83 M boric acid, 10 mM Na2 EDTA, pH 8.3). Swirl to mix.
- Add 60 µL of 10% (w/v) ammonium persulfate and 5 µL of TEMED. Mix thoroughly.
- Immediately pour the gel mixture into the gel cassette (Mini-Protean II, BioRad Co, Richmond, CA) (0.75 mm thick; 8 X 10 cm).
- Insert the comb at the top of the gel. Let the gel solidify for 20 min.
- Add 1 µL of the loading buffer to 2.5 µL of the final, amplified reaction mix.
- Load this sample into the gel and conduct electrophoresis at 200 v.
- Stop the electrophoresis when the front of the dye migrates to the bottom of the gel.
Silver Staining for DNA visualization
Gels were silver stained using a modified procedure of Bassam et al. (1991) :
- Gently shake the gel in 7.5% (v/v) glacial acetic acid for 10 min at room temp.
- Rinse the gel in deionized water twice for about 2 min each.
- Incubate the gel in 10% oxidizer solution (Bio-Rad #161-0444) for 5-10 min.
- Rinse the gel in water three times for about 5 min each. Use fresh deionized water each time.
- Immerse the gel in silver staining solution (100 mg silver nitrate and 150 µL formaldehyde in 100 mL water) for 20 min.
- Pour out the silver stain solution, and wash the gel quickly with deionized water.
- Immerse the gel in an ice-cold developer solution (8C) (3 g sodium carbonate, 300 µL formaldehyde, and 200 µg sodium thiosulfate in 100 mL water) until optimal image intensity is obtained.
- Stop the developing process by immersing the gel in 7.5% ice-cold glacial acetic acid.
- Air dry the gel and back it with a GelBond plastic film (FMC BioProducts, Rockland, ME).
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