实验10 除去RNA中的基因组DNA.pptVIP

* 实验12 除去RNA中的基因组DNA DNase I（RNase Free） TaKaRa Code： D2215 1. 在微量离心管中配制下列反应液，全量50 μl。 全RNA 20～50 μg 10×DNase I Buffer 5 μl DNase I（RNase-free，5 U/μl） 2 μl RNase Inhibitor（40 U/μl） 0.5 μ DEPC H2O up to 50 μl 2. 37℃反应20～30分钟。 3. 使用以下两种方法使DNase I失活。 A．热处理 (√) （1）加入2.5 μl 0.5 M EDTA 80℃ 2 min。 （2）用RNase Free dH2O定容至100 μl。 B．苯酚/氯仿抽提 （1）用50 μl RNase Free dH2O定容至100 μl后加入等体积的苯酚/氯仿/异戊醇（25：24：1）混匀。 （2）室温，13,500 rpm 5 min离心，取上清。 （3）加入等体积的氯仿/异戊醇（24：1）混匀。 （4）室温，13,500 rpm 5 min离心，取上清。 4. 加入10 μl 3 M醋酸钠，250 μl冷乙醇，冰上放置10 min。 5. 4℃，13,500 rpm 15 min离心，弃上清。 6. 加入70%冷乙醇洗净，4℃，13,500 rpm 5 min离心，弃上清。 7. 沉淀干燥。 8. 用适量的RNase Free dH2O溶解. 9. PCR检测确认是否除去基因组DNA. Promega RQ1 RNase-Free DNase (M610A) Description: RQ1 (RNA-Qualified) RNase-Free DNase is a DNase I (endonuclease) that degrades both double-stranded and single-stranded DNA, producing 3′-OH oligonucleotides (1). (RQ1 RNase-Free DNase may be used in applications where maintaining the integrity of the RNA is critical.) This DNase is suited for applications such as nick translation (2), production of random fragments (3), cleavage of genomic DNA for footprinting (3), removal of DNA template after in vitro transcription (4), and removal of DNA from RNA samples prior to applications such as RT-PCR (5). In the presence of Mg2+, DNase I attacks each strand of DNA independently, and the sites of cleavage are distributed in a statistically random fashion (6). In the presence of Mn2+, DNase I cleaves both strands of DNA at approximately the same site to yield fragments with blunt ends or protruding termini of one or two nucleotides in length (6). 10X Reaction Buffer (M198A): The RQ1 DNase 10X Reaction Buffer provided with this enzyme has a composition of 400mM Tris-HCl (pH 8.0), 100mM MgSO4 and 10mM CaCl2. Enzyme Storage Buffer: RQ1 DNase is supplied in 10mM HEPES (pH 7.5), 50% glycerol (v/v), 10mM CaCl2 and 10mM MgCl2. Heat Inactivation: 10 minutes at 65°C in the presence of Stop Solution. Inhibitors: EGTA; EDTA