DNALaddering
I. Protocol1. Harvest cellsOptional: wash plate with 37°C PBS (gently, so as not to lose cells); check on microscope,after aspirate PBS or mediaPlace plate on iceLyse with DNA ladder buffer 4°C150 ul/60 mm plateScrape, transfer to eppendorfRotate 4°C 20 min2. Microcentrifuge 15 min, 4°CTo pellet chromatinTransfer sup3. Phenol/CHCl3 extractAdd equal volume phenol/CHCl3Mix by pipettingMicrofuge 2 min; transfer supWill ......阅读全文
DNA Laddering
I. Protocol1. Harvest cellsOptional: wash plate with 37°C PBS (gently, so as not to lose cells); check on microscope,after aspirate PBS or mediaPlace
DNA laddering assay for treated cells
Characteristics of this procedure: I found the procedure described by Gong et al. to be a convenient and successful method to detect DNA ladderin
Staining Methods for cell death Z. Xia 10/2/95
The simplest way: trypan blue. Dead cells stain blueNon-fixed cells: FDA(fluorescein diacetate)-green, alive cells; P.I. (propidium iodide)-red, dead
Staining Methods for cell death
The simplest way: trypan blue. Dead cells stain blue Non-fixed cells: FDA(fluorescein diacetate)-green, alive cells; P.I. (propidium iodide)-r
Staining Methods for cell
death Z. Xia 10/2/95 The simplest way: trypan blue. Dead cells stain blue Non-fixed cells: FDA(fluorescein diacetate)-green, alive cells;
Cell-free System for the examination of apoptotic activity
IntroductionIn our lab we use the term 'cell-free system' when we talk about the examination of apoptotic activity in cytoplasmic extracts. T
DNA重组（DNA recombination）技术：DNA序列测定-2
目前应用的两种快速序列测定技术是Sanger等（1977）提出的酶法（双脱氧链终止法）和Maxam（1977）提出的化学降解法。虽然其原理大相径庭，但这两种方法都同样生成相互独立的若干组带放射性标记的寡核苷酸，每组核苷酸都有共同的起点，却随机终止于一种（或多种）特定的残基，形成一系列以某一特定核苷酸
DNA重组（DNA recombination）技术：DNA序列测定-3
2．利用末端转移酶和α-32P-ddNTP标记DNA的3ˊ-末端 在二价阳离子存在下，末端转移酶催化dNTP加入DNA分子的3ˊ-羟基末端，如果作为底物的核苷酸经过修饰（如ddNTP），则可以在DNA的3ˊ-OH上仅加入一个核苷酸。对于双链DNA片段，亦存在DNA的两侧均被标记问题，可通过上述同
DNA重组（DNA recombination）技术：DNA序列测定-1
㈣ DNA聚合酶 如前所述，选用合适的DNA聚合酶进行测序反应也是保证测序质量的重要因素之一。常用于双脱氧末端终止法测序的有几种不同的酶： 1．大肠杆菌DNA聚合酶Ⅰ大片段（Klenow片段） 此酶是最早用于建立Sanger测序的酶。但通常会有两个问题：①Klenow片段的持续合成能力较
DNA重组技术(DNA Recombination)
一、DNA 的酶切与连接（1）酶切反应：同质粒DNA 的鉴定，只不过是质粒DNA 换为载体DNA 。若大量酶切，则成比例增加。（2）加2倍体积的预冷无水乙醇和1/10体积的3mol/l NaAc混匀，-20℃2h以上。（3）15000rpm离心15min，弃上清。（4）加入75%乙醇洗涤2次，离心弃