DNA重组

Prepare a ligation mix:Ligation Mix (2x)10x ligase buffer1.0 mldigested vector(0.1 mg/ml)1.0 mlH2O6.0 mltotal8.0 mldivide ligation mix into two Eppendorf tubesLigation Rxn InsertControlligation mix ...

Cohesive End Ligation1) The ligation mixture contains the following: vector DNA (~100 ng) insert DNA (equimolar or 2 or 3 X molar concentration of vector) water added to 18 µl 2) Heat the mixture at 4 ...

set up the following reaction:CIP Rxn H2O7.8 ml10x cip rxn buffer2.0 mlDNA(e.g; 3 kb vector; 0.2 mg/ml; 2 mg total)10.0 ml(1 u/ml) CIP0.2 mltotal20.0 mlincubate for 60 min at 37°C add 1.14 ml 0.2 M ...

DEPHOSPHORYLATION OF LINEARIZED DNAALKALINE PHOSPATASE(CIP) DIGESTION Digestion of protruding 5'-ends1. Precipitate digested DNA and resuspend in a 100ml of 1X CIP Buffer.2. Add 1U CIP for 100pmol 5'- ...

DEPHOSPHORYLATION OF LINEARIZED DNAALKALINE PHOSPATASE(CIP) DIGESTION Digestion of protruding 5'-ends1. Precipitate digested DNA and resuspend in a 100ml of 1X CIP Buffer.2. Add 1U CIP for 100pmol 5'- ...

Phosphatasing with Shrimp Alkaline Phosphatase (S.A.P.)Use to prevent linearized vector plasmid from recircularizing. The phosphatase from shrimp is easier to get rid of than is CIP because SAP is hea ...

Template PreparationThe quality of sequencing results is directlyrelated to the quality of the template. ABI recommends a minialkaline-lysis/PEG precipitation procedure (the Core can supplyinformation ...

Interpreting DNA Sequencing ResultsEvaluating ChromatogramsMany problems with sequencing results are not recognized by viewing the text file alone. Therefore the quality of your sequence should alway ...

The following dye-labeled terminator reaction chemistries have been designed to balance conservation of reagents with the resulting sequence product signal strength. When coupled with BACPAC Resource ...

Preparation of DNA Template For Direct Sequencing of Large Insert PAC and BACPlasmidundefined This protocol is designed to allow the use of 96-well trays (8x12 microtiter tray format) and multi-channel de ...

Preparation of DNA Template For Direct Sequencing of Large Insert PAC and BACPlasmidundefined This protocol is designed to allow the use of 96-well trays (8x12 microtiter tray format) and multi-channel de ...

MATERIALS: 2-glass plates 1 sharks -tooth comb and spacers Whatman 3 mm paper 30 or 40% acrylamide-bis (19:1) 10X TBE urea 10% ammonium persulfate TEMED 60 cc. syringe Rain-ex Preparation of glass pla ...

MATERIALS: 2-glass plates 1 sharks -tooth comb and spacers Whatman 3 mm paper 30 or 40% acrylamide-bis (19:1) 10X TBE urea 10% ammonium persulfate TEMED 60 cc. syringe Rain-ex Preparation of glass pla ...

DNA Sequencing GelsBuffers and gel solutionsLong Ranger: we started using this in early 1995. Great stuff; the best thing is that the gels are not sticky after drying even without removal of the urea. ...

I have been using an alternative to wedge gels that saves acrylamide cuts gel drying time to 20 min and gives as good or better band squashing at the bottom of the gel. It was published in BioTechniqu ...

生物通报道： EZ1病毒迷你试剂盒（EZ1 Virus Mini Kit）是一种能够用于生命科学研究中的病毒DNA和RNA提纯的新型试剂盒。这种试剂盒提供了一种全自动的同步纯化来自血清、血浆和无细胞体液中的病毒DNA和RNA的能力，并且能对下游分析提供高灵敏度的检测。BioRobot® EZ1工作区的自动化使研究人员能够一次完成1到6个样品的处理。高产出的特点（即使在低的病毒浓度下）使下游检测具有 ...

一、质粒DNA的提取及鉴定（一）质粒DNA的提取及鉴定　　1．收获细菌　　（1）将2ml含相应抗生素的LB液体培养基加入到通气良好的15ml的试管中，接入一单菌落，于37℃剧烈振荡培养过夜。　　（2）将1.5ml培养物倒入1.5ml离心管中，用台式离心机于4℃以12000g离心5min，将剩余的培养物贮存于4℃。　　（3）吸尽培养液，使细菌沉淀尽可能干燥。　　2．碱裂解法小量抽提质粒　　（1）将细 ...

DNA的重组　　（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，离心弃上清，真空抽干。　　（5）加入适量1×TE溶解。如此可得线性化载体DNA ...

相关基础知识 1.DNA片段的纯化： DNA纯化的主要目的是回收得到纯的目的DNA片段，去除影响DNA连接酶活性的物质以及其它的DNA片段。纯化DNA片段的方法有多种，如：电洗脱法、从低熔点或普通琼脂糖凝胶中回收、玻璃珠纯化、柱层析以及硅胶吸附等方法。目前一些生物公司推出了直接从 PCR产物中或琼脂糖凝胶中回收DNA片段的试剂盒，有效地加快了DNA的纯化的速度。 2.DNA重组 DNA重组本 ...

DNA是遗传信息的载体是最重要的生物信息分子是分子生物学研究的主要对象因此DNA的提取也应是分子生物学实验技术中的最重要、最基本的操作，如不能有效的完成DNA提取方面的工作，那就根本谈不上进行分子生物学方面的实验。在DNA提取过程中应做到1根据不同研究需要，保证结构的相应完整性；2尽量排除其它大分子成分的污染(蛋白质、多糖及RNA等)；3保证提取样品中不含对酶有抑制作用的有机溶剂及高浓度的 ...