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1. Pick single colony and inoculate 250 ml of LB broth containing 100 m g/l ampicillin or appropriate antibiotic. Shake at 250 RPM overnight.2. Centrifuge cells in a Sovall GSA (250 ml)or SLA-3000 (500 ml) rotor at 5 k × g for 10 minutes.3. Resuspend cell pellet in 5 ml of GTE buffer (50 mM Glucose, 25 mM Tris-Cl, 10 mM EDTA, pH 8) by ...

Restriction digests consist of: 15.75 ml ddH2 O 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℃ 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℃ if necessary. Prepare 1% agarose gels in 1X TAE: Cool molten agarose to 46℃ in a water bath with occasional stirring. Pour into 20X25 cm UV tra

Materials: RPMI 1640 medium fetal calf serum (FCS) 20% Colcemid (e.g. Boehringer Mannheim cell biology reagents Best.-Nr. 295892) cell cuture flask Phythemaglutinin PHA-L (Seromed M 5030) CO2 ce ...

Introduction This manual is a compilation of many of the everyday methods used in the average molecular biology laboratory with emphasis on the techniques for large scale DNA sequencing protocols and ...

This is a very fast mini-prep protocol which is suitable for sequence analysis and restriction digests. Although the yield is higher than Protocol D.1, there is considerable chromosomal DNA, RNA and protein contamination. Solutions Sucrose/Tris 25% sucrose 25 g sucrose 50 mM Tris pH 8.0 5 ml 1M Tris pH 8.0 up to 100 ml with Q store at room temperature Triton Lysing Mix 5% Triton X-100 5 ml Triton X-100 5% sucrose 5 g sucrose 50 mM Tris pH 7.5 5 ml 1M Tris pH 7.5 50 mM EDTA 10 ml 0.5 M EDTA pH 8.

Solutions Gel Stocks Diluent 5X Buffer 25% Acrylamide 209 g Urea 209 g Urea 209 g Urea up to 500 ml Q 250 ml 10X TBE 120.8 g Acrylamide up to 500 ml Q 4.1 g BIS up to 500 ml Q 2.5 M NH4 OAc 19.2 g NH4 OAc up to 100 ml Q Formamide Dye 9 ml deionized formamide 500 m l 10X TBE 500 m l 0.2% bromophenyl blue and 0.2% xylene cyanol Other Reagents Needed: 0.22 m M disposable syringe filters, short wave UV source, intensifying screen for UV shadowing Procedure • Pour a 20% denaturing gel: 12 ml 5X Buffe

T-RFLP（末端限制性片段长度多态性）该技术在应用的过程中，肯定需要在实验条件上不断进行改进，而这种改进的好坏自然而然需要实验结果的验证。 V. Grüntzig在2002年做了该工作的一部分，结果认为，在限制性酶切时，很有必要去除其中影响DNA的酶切过程，并且实验证明了具体的酶切时间。 具有说服力的结果如下： 1、T-RFLP出数据的速度快，不过只是具有半定量性； 2、DNA片断的分 ...

This protocol was designed to generate directionally end-labeled probes for DNaseI footprinting but it can be used for any application that requires end-labeled DNA probes. Solutions 10 mM dNTP Stocks Thaw100 mM stocks (NEB or Boehringer Mannheim) on ice and dilute 10-fold in Q. store small (10-20 ml) aliquotes at -80 degrees and thaw on ice just prior to use 10 X Klenow Buffer 0.5 M Tris 7.5 500 ml 1 M Tris 7.5 0.1 M MgCl2 100 ml 1 M MgCl2 10 mM DTT 100 ml 0.1 M DTT 0.5 mg/ml BSA 50 ml 10 mg/ml

Glass wool method: Run TAE agarose gel and cut the appropriate band out with a clean razor blade. Poke a small hole with hot needle on the bottom of an eppendorf tube, and jam the hole with a little of siliconized glass wool. Put the gel in the tube and put this tube onto another tube and spin with 5-7 Krpm for 5-10 min. Add 1/2 vol. of phenol and 1/10 vol. of 3 M NaAc of the gel volume to the agarose tube and spin for another 5 min. Discard the agarose tube. Add 1/2 vol. of chloroform to th

Materials: • 0.8 % agarose gel in 1x TAE • Digested DNA • Glass Milk • NaI solution • New Wash Procedure: 1) Run digested DNA out on agarose gel slowly (70 V on BioRad gel) 2) Use long wave UV lamp to visualize bands. Cut out band with scalpel. Cut smallest possible piece. 3) Put gel slice in an eppendorf tube and weigh to figure out volume of gel slice. (empty tube approx. 1 g). 4) Add 3 vol of NaI solution (gel slice is usually ~200 mg, therefore, add 600 µl NaI). 5) Melt gel slice in 55℃ wate

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（一）第一向等电聚焦 1. 从冰箱中取-20℃冷冻保存的水化上样缓冲液（I）（不含DTT，不含Bio-Lyte）一小管（1ml/管），置室温溶解。 2. 在小管中加入0.01g DTT， Bio-Lyte 4-6、5-7各2.5ml，充分混匀。 3. 从小管中取出400ml水化上样缓冲液，加入100ml样品，充分混匀。 4. 从冰箱中取-20℃冷冻保存的IPG预制胶条（17cm pH 4-7），室温中放置10分钟。 5. 沿着聚焦盘或水化盘中槽的边缘至左而右线性加入样品。在槽两端各1cm左右不要加样，中间的样品液一定要连贯。注意：不要产生气泡。否则影响到胶条中蛋白质的分布。 6. 当所有的蛋白质样品都已经加入到聚焦盘或水化盘中后，用镊子轻轻的去除预制IPG胶条上的保护层。 7. 分清胶条的正负极，轻轻地将IPG胶条胶面朝下置于聚焦盘或水化盘中样品溶液上，使得胶条的正极（标有+）对应于聚焦盘的正极。确保胶条与电极紧密接触。不要使样品溶液弄到胶条背面的塑料支撑膜上，因为这些溶液不会被胶条吸收。同样还要注意不使胶条下面的溶液产生气泡。如果已经产生气泡，用镊子轻轻地提起胶条的一端，上下移动胶

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转染技术的选择对转染结果影响也很大，许多转染方法需要优化DNA与转染试剂比例，细胞数量，培养及检测时间等。一些传统的转染技术，如DEAE右旋糖苷法，磷酸钙法，电穿孔法，脂质体法各有利弊，其主要原理及应用特点见下表：

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第一节 概 述 把一个有用的目的DNA片段通过重组DNA技术，送进受体细胞中去进行繁殖和表达的工具叫载体(Vector)。细菌质粒是重组DNA技术中常用的载体。 质粒(Plasmid)是一种染色体外的稳定遗传因子，大小从1-200kb不等，为双链、闭环的DNA分子，并以超螺旋状态存在于宿主细胞中。质粒主要发现于细菌、放线菌和真菌细胞中，它具有自主复制和转录能力，能在子代细胞中保持恒定的拷贝数， ...

一、导论 已经提出过许多方法用于从细菌中提纯质粒DNA ， 这些方法都含有以下3个步骤： 细菌培养物的生长。 细菌的收获和裂解 质粒DNA 的纯化。 （一）细菌培养物的生长 从琼脂平板上挑取一个单菌落，接种到培养物中( 有含有行当抗生素的液体培养基中生长) ，然后从中纯化质粒，质粒的提纯几乎总是如此。现在使用的许多质粒载体（如pUC 系列）都能复制到很高的拷贝数，惟致只要将培养物放在标准LB 培养 ...

小于100bp的片断通常在电泳时就比较难观察分辨，需要用分辨率很高的琼脂糖或者丙烯酰胺凝胶。比如Cambrex的NuSieve 3:1或者GTG，或者是大名鼎鼎的MetaPhor琼脂糖。所以实际上对于小片断，可以分为两种情况：一个是真的要回收电泳中特别小的片断，这种情况我们前面有提到，比如Qiagen MinElute系列可以回收70bp以上的片断；而QIAEX纯化介质可以回收40bp以上的小片断，可以根据情况选择。 ...

一、感受态细菌的使用： 1. 对于感受态细菌效率的测定或连接产物的转化(其它如基因突变产物的转化等参考连接产物进行)：