第六讲-琼脂糖凝胶电泳.pptVIP

琼脂糖凝胶电泳 凝胶制备 DNA Marker 加样与电泳 观察与记录 注意事项 AGAROSE 强度 600, 1000~1200, 1500 熔点 65?C, 86~88?C 凝固 26~30?C, 36~38?C 电渗 (Electroendosmosis) EEO/mr 用途：100bp~2kb Ampli Ag. 1kb~10kb Molecular Bio. Ag. 105 ~106 bp Pulsed field certified Ag. 106 bp Chromosomal DNA Ag. DNA Marker Lamda DNA/ Re 1kb, 100bp, 50, 10 bp ladder ( Molecular bio, amplification) Pulse ele. Marker Chromosomal Marker The appropriate marker matching DNA bands Melting time0.7~1.5% 5 min (Microwave oven) 无折光颗粒，均匀 冷却与凝胶 充足的时间，20min, 氢键, 网状结构 EB 0.1~0.5ug/uL(positive charge) chamber, soak TAE buffer provides faster electrophoretic migration of linear DNA and better resolution of supercoiled DNA TBE buffers have a stronger buffering capacity for longer or higher voltage electrophoresis runs. Ethidium Bromide Staining Procedure 1. Place the gel into the appropriate volume of 0.5 μg/ml ethidium bromide (EtBr) stain for 15–30 minutes. Use enough staining solution to cover the entire gel. Caution: Ethidium bromide is a suspected carcinogen and should be handled with extreme care. Always wear gloves, eye glasses, and a laboratory coat. Dispose of used EtBr solutions and gels appropriately 2. Destain the gel for 10–30 minutes in dH2O using the same volume used for staining.Note: Ethidium Bromide can be removed from the DNA with extended destaining. This will cause lower sensitivity of detection. However, insufficient destaining will create higher background fluorescence. 3. Rinse the gel briefly with dH2O to remove any residual staining solution. 4. Place the gel on a UV transilluminator for nucleic acid visualization and analysis. DNAEthidium Bromide complexes may be illuminated with UV light of 254, 302, or 366 nm. Sensitivity decreases with illumination at higher wavelengths. However, nicking of DNA will increase below 302 nm. Table 2.5 gives the percentage of transmittance of