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Protocol

RESTRICTION ENZYME DIGESTS AND SOUTHERN BLOTTING
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1. Typically 11-16 ug of genomic DNA is digested according to manufacturers recommendation with 3-5U/ug genomic DNA for 18 hours. At initiation of digest 1 ug of genomic DNA is removed from the total digest (which includes enzyme) and is mixed with 0.5 ug-1 ug of test DNA. Typically pBR322, or some other plasmid whose restriction pattern may be known and that can be used to determine the extent of digestion. 2. After 18 hrs the test digestion is stopped with 2mM EDTA, 10 x Sample Running Buffer (50% glycerol, 0.02% Bromephenol blue) is added to sample to make 1X. The samples are heated for 51 at 600C and chilled and loaded onto gel. Test gels are routinely run 100V for 3.5 hrs. If test DNA looks completely cleaved we stop Rxtn w/2mM EDTA, evaporate sample and prepare for gel anaylsis. 3. gel - 0.7% agarose lx TAE or 1 x TGA, or 1 x TBE (or 2x TBE) with gel and well size dependent on number and volume of samples. 1 x TBE = 0.89M TRIS-borate 0.89M Boric acid, lmM EDTA Gels are run in 1 x TBE or 2 x TBE Running Buffer (or 1 x TAE or 1 x TGA) for either 350 V. hrs (35 volts for 10 hrs or 100 volts for 3.5 hrs - 4).
PREPARE FOR SOUTHERN BLOT 1. remove gel, photograph using a UV box 2. soak gel in 0.4N NAOH 3. set up blot in 0.4N NAOH as pictured on next page 4. cut Zetaprobe membrane (BioRad) to size of gel (after cutting of wells) BLOT 1. wet zetaprobe membrane 2. construction 3 cm paper towels cm blotting pads (can be paper towels) pieces Whatman 3 mm cut to slightly small than gel (1-2 mm) zeta probe gel bottom sido reservoir tank Whatmann 3mm wicks sually the gel former upside down buffer 0.4N NaO 3. blot - cover w/ saran wrap to prevent evaporation and place a light weight on top 4. neutralize gel in 5x SSC for 151 5. air dry, bake 2 hrs at 800C 6. photograph blot on UV box HYBRIDIZATION SOLUTIONS: DXSO4 - Nylon membranes i.e., NYTRAN, zetaprobe Prehybridization solution: Working Stock Dil Amt. 50% formamide 100% 1:2 5 ml 4x SSPE 20x 1:5 2 ml 1% SDS 20% 1:20 0.5 ml 25% Denhardts 50% 1:20 0.5 ml 0.5 mg/ml SSDNA lmg/ml 1:10 1.0 ml d-H20 1.5 ml 10 ml 420C 30 minutes Hybridization solution: 47% formamide 100% 4.7 ml 10% DxSO4 50% 2.0 ml 3X SSPE 20x 1.5 ml 1% SDS 20% 0.5 ml 100x Denhardts 50% 0.5 ml 0.5mg/ml SSDNA 1.0 ml 10 ml 420C 12-16 hours 2. ssDNA - use 2 separate vials if 2 separate probes. Use 2059 tubes to boil the ssDNA. Boil the probe in the ssDNA, otherwise the probe will evaporate since such a small volume. Boil 5 minutes, then quench on ice.
RESTRICTION ENZYME DIGESTS AND SOUTHERN BLOTTING

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