• 西亚试剂::RFLP和RAPD技术原理和操作步骤
  • 西亚试剂::RFLP和RAPD技术原理和操作步骤
  • DNA分子水平上的多态性检测技术是进行基因组研究的基础。RFLP(Restriction Fragment Length Polymorphism,限制片段长度多态性)已被广泛用于基因组遗传图谱构建、基因定位以及生物进化和分类的研究。RFLP是根据不同品种(个体)基因组的限制性内切酶的酶切位点碱基发生突变,或酶切位点之间发生了碱基的插入、缺失,导致酶切片段大小发生了变化,这种...详细+
  • 西亚试剂::Electrophoretic Mobility Shift Assay(EMSA)
  • 西亚试剂::Electrophoretic Mobility Shift Assay(EMSA)
  •   Electrophoretic Mobility Shift Assay (EMSA) 20cm Gel Preparation, Sample Separation, and Visualization: Glass Plate Preparation: • Wash both panes of glass with windex and 70% EtOH • Dry with KimWipe • Assemble with toothed spacers on sides, winged spacer on bottom • Clamp corners, bottom and side with binder clips • Lie on a slight angle (can use microfuge tube rack) Native PAGE Preparation: Add in ...详细+
  • 西亚试剂::GELSHIFT--胶体位移
  • 西亚试剂::GELSHIFT--胶体位移
  • )Nuclear extract: see nucprep.ptc & nucext.ptc extracts should be at least 3ug/ul Probe preparation: see endlabl.ptc & isotach.ptc. probe shouldbe 10©20k cpm/ul. Fragments larger than 400bp should not be used. Make A stock (25ug/50ul) of probe plasmid digested at one end w/ 5' overhang. 3)Binding rxn: 1-2ul probe (0.5ng or 20k cpm in isotach 40mM Tris 7.5 buffer) 1-2ul poly dIdC or dAdT (3ug/ul in NEB, sonicated to 300©...详细+
  • 西亚试剂::FOOTPRINTING WITH DNASE1
  • 西亚试剂::FOOTPRINTING WITH DNASE1
  • probe: same as used for gelshift), isolated by isotacelectrophoresis w/out EtOH ppt which can enature dsDNA 2)probe mix/rxn: volumes x # samples 1ul probe (0.1©0.5ng or 10©20kcpm) 0.15ul 20mM EDTA 0.4ul 10ug/ul dIdC or dAdT (from gel shift assay) 0.5ul H2O 3)DNAse mix: made up near end of binding incubations. DNAse l(Worthington DPFF,Cat #LS0006330, lot #58A047,5mg) is 1mg/ml in150mM NaCl, 50% glycerol, store at &co...详细+
  • 西亚试剂::EMSA using ds Oligonucleotides
  • 西亚试剂::EMSA using ds Oligonucleotides
  •   Solutions 10X Annealing Buffer 200 mM Tris 8.0 200 ml 1M Tris pH 8.0 10 mM EDTA 8.0 20 ml .5M EDTA pH 8.0 500 mM NaCl 100 ml 5M NaCl 280 ml Q store at room temperature 10X Klenow Buffer 500 mM Tris 7.5 500 ml 1M Tris pH 7.5 100 mM MgCl2 100 ml 1M MgCl2 10 mM DTT 20 ml 0.5 M DTT 0.5 mg/ml BSA 50 ml 10 mg/ml BSA (NEB) 330 ml Q store at -80° C in 50 ml aliq. ...详细+
  • 西亚试剂::Gel Shift (EMSA) Protocol
  • 西亚试剂::Gel Shift (EMSA) Protocol
  •   There are multiple variations to this protocol, but we find that this one works well in all cases we tested. Reagents: 5X EMSA Buffer: 50mM HEPES (pH 7.9) 375 mM KCl 12.5 mM MgCl2 0.5 mM EDTA 5 mM DTT 15% Ficoll 32P-labeled oligonucleotide probe polydI/dC:  1 mg/ml in TE BSA:  10 mg/ml in TE 5% Polyacrylamide gel (30 ml) 22 ml water 3 ml 5X TBE ...详细+
  • 西亚试剂::DNA fingerprinting - agarose gel
  • 西亚试剂::DNA fingerprinting - agarose gel
  • Restriction digests consist of: 15.75 ml ddH2O 1 µl 10 X buffer B (Boehringer Mannheim) 0.25 µl HindIII (40 U/µl) 3 µl DNA Set up digestions in 96 well plates. Incubate at 37°C for 4.5 hours. This can be done in a thermocycler. After digestion, a brief centrifugation will collect DNA at the bottom of wells. Seal plates with foil tape and store at 4°C if necessary. Prepare 1% agarose gels in 1X TAE: Cool ...详细+
  • 西亚试剂::DNA fingerprinting
  • 西亚试剂::DNA fingerprinting
  • 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 pH8.0 or 50 µl ddH2O Miniprep with Qiagen kit   Check the quality and quantity of miniprep BAC DNA ...详细+
  • 西亚试剂::Chemical Sequencing of DNA
  • 西亚试剂::Chemical Sequencing of DNA
  • This is a rapid method for chemical DNA sequencing which is commonly used as ladder for footprinting reactions or for sequencing of short DNA oligonucleotides. Reference: Bencini et al. (1984) Biotechniques 2: 4-5. Steve Hahn/Hahn Lab   -------------------------------------------------------------------------------- The method below works well for Sequencing of DNA of greater than ~40 bp. Typically, about 150 bases of sequenc...详细+