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The present study highlights the choice of DNA dyes for routine usage. (i) Although EB has slightly higher sensitivity for bands F100 bp, the sensitivity of SYBR Gold or SYBR Green I is already ideal for conventional PCR products because their size is always )100 bp. (ii) Extension of integration time can help the visualization of small fragments such as 50 bp with SYBR Green . (iii) SYBR Gold is better than SYBR Green I at detecting small fragments. (iv) SYBR Gold and Green, when mixed with DNA either before loading or incorporated in the gel, slow the electrophoretic mobility of DNA fragments in a manner dependent on the concentrations of the dyes and of the DNA, and thus cannot be used to accurately determine DNA fragment sizes unless they are added to the gel after electrophoresis. These results indicate that the observation of different sizes could be due either to different PCR product sizes or to different amounts of DNA for the same PCR product, whichwas the limitation of SYBR Gold or SYBR Green I. (v) GoldView has a higher background and lower sensitivity compared with SYBR-LB1, SYBR-LB2 and EB, which makes it only suitable for DNA samples of high concentrations. (vi) SYBR Gold or SYBR Green I are cost-effective and safe alternatives to EB . In our laboratory, the SYBR Gold method (i.e., 10=SYBR-LB1) is now used routinely by all members of our group with great consistency and success. Ethidium bromide (EB) is a mutagen and toxin that is widely used in the laboratory for visualizing nucleic acids. Decontamination of EB is a problem for laboratory safety . SYBR Gold (Molecular Probes, Eugene, OR, USA), SYBR Green I (Molecular Probes)and GoldView (SBS Genetech, Beijing, China) can detect nucleic acids in agarose gel electrophoresis with a sensitivity greater than that of EB (SYBR Gold and SYBR Green I) or equal to that of EB (Gold-View). More importantly, they appear to be much less mutagenic than EB, based on Ames mutagenicity tests. The manufacturer of GoldView, a little more expensive than EB, recommends it for routine use in methods similar to those for EB. SYBR Gold and SYBR Green I are costly reagents, but by adding them into the loading buffer it is possible to reduce their cost while maintaining sensitivity at the same level as EB . 原谅我这文盲吧  |
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落迦(金币+10,VIP+0):谢谢
落迦(金币+10,VIP+0):谢谢
| 本研究重点研究了DNA常规染色剂的筛选。(i)尽管EB对于F100bp条带的灵敏度稍高，SYBR Gold或SYBR Green的灵敏度对于常规聚合酶链反应产品来说已经足够理想，因为它们的大小一般为100bp。(ii)积分时间的延长有助于增强被SYBR Green染色的小片段如50bp的可适化。(iii) 对于检测小片段来说，SYBR Gold比SYBR Green I更佳。(iv) 在加载或混入明胶前将SYBR Gold和SYBR Green与DNA混合能够降低DNA片段的电泳迁移率，并且该影响与染色剂及DNA的浓度有关。因此，除非在电泳后加入明胶，否则不能用于精确测定DNA片段大小。上述结论表明，观察到的不同大小可能是由于不同聚合酶链反应产品的粒度不同，或者对于相同聚合酶链反应产品的不同DNA量，而后者是SYBR Gold或 SYBR Green I的局限性。(v)相比于SYBR-LB1, SYBR-LB2和EB，GoldView的背景噪声更高且灵敏度更低，这使得其只适用于高浓度DNA样品。(vi) SYBR Gold 或SYBR Green I的价格便宜且安全，可以作为EB的替代品。 |
2楼2008-05-14 15:08:14
★
落迦(金币+1,VIP+0):高手呀
落迦(金币+1,VIP+0):高手呀
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剩余的部分： 在我们实验室，现在团队所有成员都在把SYBR Gold法作为常规方法使用，并取得了非常好的稳定性和成功。溴化乙锭作为一种诱变剂和毒素在实验室被广泛应用于观察核酸。而EB的净化则是实验室净化的一个问题。SYBR Gold(Molecular Probes分子探针, Eugene, OR, USA), SYBR Green I (Molecular Probes)和GoldView (SBS Genetech, Beijing, China) 能以比EB(SYBR Gold和SYBR Green I)更高或与EB (Gold-View)相当的灵敏度检测出琼脂糖凝胶电泳中的核酸。更重要的是，Ames致突变试验表明，它们的致突变性比EB要小得多。价格比EB稍高的GoldView生产商推荐作为常规使用，方法类似于EB。SYBR Gold和SYBR Green I是较为昂贵的试剂，但是通过将它们加入到负载缓冲液中可能降低其成本而保持与EB相同的灵敏度。 |
3楼2008-05-14 16:31:12
