近10年来，现代分子生物学技术越来越广泛地被用于人类疾病研究的诸领域，为了解病理状态下基因组DNA的变化积累了新资料。目前认为，人类基因组并非人们想像的那样稳定，诸如基因重排、扩增、缺失，突瘘和DNA甲基化类型改变等时有发生，这些改变对于基因表过和调控，以及疾病过程的发展与转归等方面均具有重要意义。 医院病理科档案中积存的大量石蜡包埋组织，是一个可靠的分子生物学研究的材料来源。在石蜡切片上进行原 ...
固定于滤膜上的RNA的预杂交、杂胶及淋洗等条件，与DNA杂交的相应条件基本相同。现简述如下1）用下列两种溶液之一进行预杂交，时间1－2小时。若于42℃进行，应采用。50％甲酰胺5xSSPE2xDenhardt试剂0.1％SDS若于68℃进行，应采用：6xSSC2xDenhardt试剂0.1％SDS 2）在预杂交液中加入变性的放射性标记探针，如欲检测低丰度mRNA， 所用探针的量至少为0.1μ ...
目前应用的两种快速序列测定技术是Sanger等（1977）提出的酶法及Maxam和Gilbert(1977)提出的化学降解法。虽然其原理大相径庭，但这两种方法都是同样生成互相独立的若干组带放射性标记的寡核苷酸，每组寡核苷酸都有固定的起点，但却随机终止于特定的一种或者多种残基上。由于DNA上的每一个碱基出现在可变终止端的机会均等，因些上述每一组产物都是一些寡核苷酸混合物，这些寡核苷酸的长度由某一种特 ...
目前应用的两种快速序列测定技术是Sanger等（1977）提出的酶法及Maxam和Gilbert(1977)提出的化学降解法。虽然其原理大相径庭，但这两种方法都是同样生成互相独立的若干组带放射性标记的寡核苷酸，每组寡核苷酸都有固定的起点，但却随机终止于特定的一种或者多种残基上。由于DNA上的每一个碱基出现在可变终止端的机会均等，因些上述每一组产物都是一些寡核苷酸混合物，这些寡核苷酸的长度由某一种特 ...
PCR amplification The PCR reaction mix (10 µL) contains template DNA (2 ng/µL) primer (0.3 µM) Taq DNA polymerase Stoffel Fragment (Perkin Elmer; 5 U) Mg++ (2.5 mM) buffer (1X) and overlaid with a dro ...
BAC DNA preparation Miniprep with AutoGen 740 Innoculate 3-4 ml LB with 20µg/ml chloramphenicol Grow at 37 °C overnight with shaking Isolate BAC DNA with AutoGen 740 Resuspend BAC DNA in 50 µl TE pH8. ...
IntroductionIncreasing evidence in literature reveals that the organization of chromosomal domains in the cell nucleus plays an important role in gene expression during cellular differentiation and de ...
IntroductionSequencing of sodium-bisulfite modified genomic DNA originally introduced by M. Frommer (Frommer et al. 1992) is a widely used “gold standard” method for DNA-methylation analysis. Since th ...
Introduction(based on Olek et al. 1996 Schoenherr et al. 2003)DNA methylation is a stable epigenetic mark which can mediate gene silencing. Bisulfite sequencing allows for precise identification of me ...
IntroductionGenomic DNA methylation is one of the most important epigenetic modifications in eukaryotes. It is essential for life and its alteration is often associated with disease. In animals most o ...
IntroductionMethylation of cytosines can mediate epigenetic gene silencing and is the most prominent DNA modification in eukaryotes. MeDIP is an immunocapturing approach to enrich DNA that is methylat ...
IntroductionNucleosomal positioning plays a pivotal role in the regulation of transcriptional initiation. Transcriptional co-activator complexes interact with nucleosomes (4) to induce nucleosomal re ...
IntroductionThe following "bisulfite genomic sequencing" protocol has been optimized for the determination of DNA cytosine methylation at transgene loci in plants. The conditions used are based on the ...
IntroductionEfficient duplication of eukaryotic genomes relies on the sequential activation of thousands of replication origins distributed along the chromosomes. This process follows a complex spatia ...
Collect fresh tissue and store at -20°C. Grind leaf tissue to a fine powder with liquid nitrogen in a prechilled mortar . Transfer ground tissue with a chilled paintbrush into a well-chilled 50-ml pol ...
DNA Digestion Make a cocktail as follows: Southern hybridization cocktail 10 X buffer3.50 µLBSA 0.35 µLddH205.65 µLRNase 0.50 mLEcoRI 10.00 µLTOTAL20.00 µL/tubeAdd 15 μL DNA to each tube containing 2 ...
Prehybridization Set hybridization oven to 65°C. Thaw 32P isotope in hood. Place membrane in hybridization tube. Add 50 mL of prehybridization buffer to each tube. Prehybridization buffer ddH20125 mL ...
Hybridization Hybridization buffer ddH2025.0 mL20X SSPE12.0 mL100X Denhardt's solution2.0 mL20% SDS1.0 mLSalmon sperm DNA 0.2 mLdextran sulfate2.0 gTOTAL40.2 mLReplace prehybridization buffer in each ...
New D14 primers.Lr21L:CGC TTT TAC CGA GAT TGG TC Lr21R:TCT GGT ATC TCA CGA AGC CTT Lr21: 669 bplr21-Fielder: 757 bplr21-Wichita: 774 bpRecipe (in 25 µl reaction). Buffer (10X)2.5 µl2.5 mM dNTP2.5 µl25 ...
New D14 primers.Lr21L:CGC TTT TAC CGA GAT TGG TC Lr21R:TCT GGT ATC TCA CGA AGC CTT Lr21: 669 bplr21-Fielder: 757 bplr21-Wichita: 774 bpRecipe (in 25 µl reaction). Buffer (10X)2.5 µl2.5 mM dNTP2.5 µl25 ...
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