电泳技术介绍.ppt

核酸的染色 RNA染色法 焦宁Y：其对RNA染色效果好，灵敏度高。脱色后凝胶本底颜色浅而RNA色带稳定，抗光且不易褪色。此染料最适浓度为0.5%。低于0.5%则RNA色带较浅；高于0.5% 并不能增加对RNA染色效果。 甲苯胺蓝O：最适浓度为0.7%，染色效果较焦宁Y稍差。 次甲基蓝：染色效果不如焦宁Y和甲苯胺蓝O，检出灵敏度较差，一般在5μg以上；染色后RNA条带宽，且不稳定，时间长，易褪色。但次甲基蓝易得到，溶解性 能好，所以较常用。 * Aside: plate theory N = # of theoretical plates = (tR/??)2 = (tR/2.354 W1/2 )2 gives information about the separation efficiency For CE, N 10,000 is routine; N 100,000 is not too difficult N 106 has been reported in ideal cases * Recap, vep (cm/s) = ?ep (cm2/V●s) x E (V/cm) Velocity mobility applied electric field The applied electric field exerts a constant accelerating force that is opposed by a frictional force supplied by the medium. The mobility ?ep = q (charge) / f (hydrodynamic drag) for spherical particles, ?ep ~ k(z/m?) Capillaries (Jorgensen 1982) 1. Dimensions 360 ?m fused silica; 50 ?m I.D. and 12 ?m thick polyimide coating (flexibility) ? Why such a small capillary? A- small capillary provides high resistance (when filled with buffer), higher R means a higher applied V w/o joule heating effects, and a higher V will lead to a more “efficient”, faster separations and narrower peaks. Joule heating occurs when electric current passes thru a conductive electrolyte buffer, this leads to a convective flow at the edges of a gel due to temperature gradient across the gel. You see evidence of joule heating at high V in gel electrophoresis as band broadening or “smiling”. It can be disruptive to an experiment due to loss of separation resolution and/or thermal denaturation of the analytes. It can be minimized or eliminated by1) lowering the E (but this will be less efficient, slows down the experiment), 2) decreasing the conductivity of the buffer (also decreases the efficiency or 3) using small diameter capillaries or thing gels so that the heat will dissipate quicker due to increased surface/volume Small sample volumes 50 ?M 20 nL/cm Detection LIMITED technique. 0.05 mm o