一、 建立一个标准的酶切反应目前大多数研究者遵循一条规则,即10个单位的内切酶可以切割1μg不同来源和纯度的DNA。通常,一个50μl的反应体系中,1μl的酶在1X NEBuffer终浓度及相应温度条件下反应1小时即可降解1μg已纯化好的DNA。如果加入更多的酶,则可相应缩短反应时间;如果减少酶的用量,对许多酶来说,相应延长反应时间(不超过16小时)也可完全反应。二、 选择正确的酶不言而喻,选择的酶在底物DNA上必须至少有 ...
Preparation of BAC (Bacterial Artificial Chromosome) DNA with CONCERT ™ High Purity Plasmid Purification System1 Courtesy Lisha Xu and Alice C. Young Molecular and Cell Biology Research and Deve ...
第一节 概 述 质粒具有稳定可靠和操作简便的优点。如果要克隆 较小的DNA 片段(<10kb)且结构简单质粒要比其它任何载体都要好。在质粒载体上进行克隆 从原理上说是很简单的,先用限制性内切酶切割质粒DNA 和目的DNA 片段 然后体外使两者相连接 再用所得到重组质粒转化细菌即可完成。但在实际工作中 如何区分插入有外源DNA 的重组质粒和无插入而自身环化的载体分子是较为困难的。通过调整连接反应中 ...
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Important : Extract the DNA within one week of receiving samples. Samples that have been processed should be frozen to prevent degradation. Freeze samples between use each day. 1. Add 600 m l of 50 mM NaOH to a 1.5 ml eppendorf tube and insert brush in tube. (Clip off handle of brush with wire cutters so tube can be closed. Cutters should be rinsed with EtOH between samples to prevent contamination.) 2. Vortex thoroughly to mix, for at least 10 s. 3. Heat to 95℃ for 5 min. 4. Spin briefly to poo
The overall sequence of events is: • Titer and plate out phage • Lift plaques onto filters and prepare them for screening • Make a probe • Hybridize the probe to the filters &b ...
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1取5ml噬菌体溶液放入无菌干净的小烧杯中,缓缓加入固体NaCl,使其终浓度为1M(0.292g)充分溶解后冰浴1小时。 24℃下11000rpm离心10min,取上清液于另一个干净烧杯中,将PEG6000加入烧杯中使其终浓度为10%W/V约0.5克),磁力棒缓慢搅拌,冰水浴1.5小时,于4℃,15000rpm离心15min,弃上清。 3将沉淀溶于300μlTE(pH8.0),加等体积的酚/ ...
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 ...
1.Obtain 65-100 µl of blood by retro-orbital bleed with a heparinized microcapillary tube. Expel blood immediately into a 1.5 ml microfuge tube containing 20 µl of 10 mM EDTA. Mix immediat ...
Restriction Enzyme Buffer Most enzymes can use REact buffers; however, some are made up separately. Use fresh Milli-Q water , siliconized or sterile glassware or disposable plastic ware to make the following stock solutions up fresh (discard after use), combine to make 5 ml of 10X reaction buffer ...
Linker Ligation (with T4 ) DNA Ligase In a microcentrifuge tube prepare a solution of blunt ended, dephosphorylated DNA (100-500ng) in TE buffer (5-7µl). Add 1-2µg of phosphorylated linkers in 5µl of TE buffer. Add: 10X ligation buffer 2µl, 50% PEG 4000 solution 2µl, deionized water to 20µl, T4 2u. Vortex the tube and spin down in a microcentrifuge for 3-5 seconds. DNA Ligase Incubate the mixture for 1 hour at 22℃ . Inactivate T4 DNA Ligase by heating the reaction mixture at 65℃ for 10 minut
Instructions Modified for Simpson Lab1.PCR: Perform a standard PCR reaction using taq polymerase. (Do not use tfl as the PCR product must have a 3' adenine overhang that only taq generates.) Include an 72℃ extension step for 7 to 30 minutes at the end of the reaction to ensure that all products are full length a ...
Materials: phenol:chloroform (1:1) chloroform Add an equal volume of buffer-saturated phenol:chloroform (1:1) to the DNA solution. Mix well. Most DNA solutions can be vortexed for 10 sec except for high molecular weight DNA which should be gently rocked. (If using Phase-Lock Gel, follow procedure M.1 ) Spin in a microfuge for 3 min. Carefully remove the aqueous layer to a new tube, being careful to avoid the interface. (Steps 1-4 can be repeated until an interface is no longer visible) To
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
常规片断级的选择指南: 所谓常规级,即回收片段大小介于100bp和10kb之间,在这个范畴内包含了一般的质粒或者克隆片段的回收。主流方法是柱回收试剂盒。 1. Qiaquick Gel Extraction Kit 品牌:Qiagen 回收范围:70bp-10kb 特点: 高达80%回收率 操作快速简单,3步完成70bp-10kb片段的回收 提供三色指示剂的电泳上样loading Bu ...
1. Add 400 μl of TE buffer then 400 μl of 1-butanol to the oligonucleotide glass vial. 2. Vortex well, then spin down in tabletop centrifuge for 10 minutes at ~2,000. rpm¹s. 3. Remove top, butanol layer with a sterile pipette tip and discard. 4. Add another 400 μl of 1-butanol. 5. Vortex well, then spin down as above in tabletop centrifuge. 6. Remove top, butanol layer and discard. 7. Transfer aqueous phase into a new 1.5 ml tube. 8. Dry in a speed vac for about 5 minutes to remove all of the b
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Footprinting Footprinting is a method for determining the exact DNA sequence to which a particular DNA-binding protein binds. Examples: hormone-receptor complexes that bind to their hormone response elements transcription factors that bind eukaryotic operators, enhancers, and silencers the lac repressor that shuts down the lac operon in E. coli [Discussion of the lac operon] How, for example, does one determine the DNA sequence to which the lac repressor binds? The procedure: Clon
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