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BioReagent,无菌过滤,用于显微镜 级 提供 ≥99% 纯度,适用于对基线干扰要求严格的色谱和分析工作流程。
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在色谱分析、有机合成和交叉偶联反应领域已被 204 篇同行评审文献引用。
IPTG 即 Isopropyl β-D-1-thiogalactopyranoside,异丙基-β-D-1-硫代半乳糖吡喃糖苷,常用于大肠杆菌的蓝白斑筛选 试验。宿主和质粒编码的片段虽都没有半乳糖苷酶活性,但它们同时存在时,α 片段与 ω 片段可通过 α-互补形成具有 酶活性的 β-半乳糖苷酶,由 α-互补而产生的 LacZ 细菌在诱导剂 IPTG 的作用下,在生色底物 X-Gal 存在时产生蓝色 菌落;而当外源 DNA 插入到质粒的多克隆位点后,破坏了 α 片段的编码,使得带有重组质粒的 LacZ 细菌形成白色菌落,这种重组子的筛选,称为蓝白斑筛选;此外,IPTG 它还可以作为具有 lac 或 tac 等启动子的表达载体的蛋白表达 诱导剂使用。本品经 0.22um 滤膜过滤除菌,可以直接加入培养基使用。
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操作方法:
一、蓝白斑筛选方法
1、倒平板:LB 固体培养基微波炉加热融化后或高压灭菌后冷却至 55℃ 以下,每 100ml 培养基中加入 200ul 20mg/ml 的 X-gal 溶液,40ul 100mg/ml 的 IPTG 溶液和相应的抗生素溶液混匀倒平板即可。
2、也可直接将 X-gal 和 IPTG 溶液涂布在 LB 平板表面:例如,直径 90mm 的平板(约 20ml 培养基)可以直接加入 40ul 20mg/ml 的 X-gal 溶液,8ul 100mg/ml 的 IPTG,用无菌涂布棒涂布均匀即可。
二、原核蛋白诱导表达方法
1、原核表达菌液在 37℃,200 rpm 培养,待 OD600=0.5~0.6,吸出 3ml 左右作为空白对照,剩下的菌液加入 IPTG 溶液,不同蛋白对 IPTG 浓度和摇菌温度要求不同,为获得需要量的蛋白,最佳诱导时间,温度,IPTG 浓度需实验者 优化。一般情况下 IPTG 浓度在 0.1mM-20mM 之间,1mM 是最常用的 IPTG 起始诱导浓度,也可设置不同的 IPTG 浓度梯度(例如:0.1mM,0.2mM,0.5mM,1mM,2mM,5mM,10mM,20mM)。
三、1 M IPTG 溶液配制
1、2.38g IPTG 加入去离子水定容到 10 mL,完全溶解后用 0.22um 的滤膜过滤除菌。
四、100 mg/ml IPTG 溶液配制
1、1g IPTG 加入去离子水定容到 10 mL,完全溶解后用 0.22um 的滤膜过滤除菌。
注意事项:
1、温度降低时 IPTG 溶液 (过滤除菌) 可能会有沉淀析出,如有沉淀将 IPTG 溶液放 37℃ 预热可重新溶解,也可剧烈 摇晃促进溶解。
2、不开封的 IPTG 溶液 (过滤除菌) 可在 -20℃ 保存 2 年,开封后若发现染菌,停止使用。
IPTG, full name Isopropyl β-D-1-thiogalactopyranoside, is commonly known as isopropyl β-D-1-thiogalactopyranoside, which is widely applied in the blue-white screening assay for Escherichia coli. Neither the host strain nor the fragment encoded by the plasmid alone possesses β-galactosidase activity. Nevertheless, when both coexist, the α-fragment and ω-fragment can undergo α-complementation to assemble enzymatically active β-galactosidase. When induced by IPTG and in the presence of the chromogenic substrate X-Gal, bacteria carrying the intact LacZ gene via α-complementation will form blue colonies. In contrast, insertion of exogenous foreign DNA into the multiple cloning site (MCS) of the plasmid disrupts the coding sequence of the α-fragment. As a result, bacterial cells harboring recombinant plasmids with inserted target DNA yield white colonies. This screening method for recombinant clones is defined as blue-white screening.In addition, IPTG serves as an inducer for protein expression from expression vectors driven by promoters such as lac or tac. This product is sterilized by filtration through a 0.22 μm membrane filter and can be directly added to culture media for use.
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Operating Protocols:
I. Protocol for Blue-White Screening
1. Pour agar plates: Melt LB solid medium in a microwave oven or cool autoclaved medium to below 55 °C. Add 200 μL of 20 mg/mL X-gal solution, 40 μL of 100 mg/mL IPTG solution and the corresponding antibiotic solution to every 100 mL medium, mix thoroughly and pour plates.
2. Alternative method: Spread X-gal and IPTG directly onto the surface of pre-poured LB plates. For a 90 mm-diameter plate (containing approximately 20 mL medium), add 40 μL of 20 mg/mL X-gal solution and 8 μL of 100 mg/mL IPTG solution, then spread evenly with a sterile spreader.
II. Protocol for Prokaryotic Protein Induction and Expression
1. Culture the prokaryotic expression strain at 37 °C with shaking at 200 rpm until the OD₆₀₀ reaches 0.5–0.6. Transfer roughly 3 mL bacterial suspension as blank control, then add IPTG solution to the remaining culture.Optimal IPTG concentration, incubation temperature and induction duration vary for different target proteins and must be optimized empirically to obtain sufficient target protein.The routinely used IPTG concentration ranges from 0.1 mM to 20 mM, with 1 mM as the standard initial induction concentration. A concentration gradient can be set as follows: 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM, 20 mM.
III. Preparation of 1 M IPTG Stock Solution
1. Dissolve 2.38 g IPTG in deionized water and bring the final volume to 10 mL. After complete dissolution, sterilize the solution by filtration with a 0.22 μm filter membrane.
IV. Preparation of 100 mg/mL IPTG Stock Solution
1Dissolve 1 g IPTG in deionized water and adjust the total volume to 10 mL. Once fully dissolved, perform sterile filtration using a 0.22 μm filter membrane.
Precautions
1. Precipitation may occur in filter-sterilized IPTG solution when the temperature drops. If precipitate appears, place the IPTG solution in a 37 ℃ environment for preheating to redissolve the sediment, or shake vigorously to facilitate dissolution.
2. Unopened filter-sterilized IPTG solution can be stored at -20 ℃ for 2 years. Discontinue use immediately if bacterial contamination is observed after opening.
H315: 引起皮肤刺激
H319: 引起严重眼睛刺激
H335: 可能引起呼吸道刺激
H351: 怀疑引起遗传缺陷
H302: 吞食有害
H312: 皮肤接触有害
H332: 吸入有害
P261: 避免吸入灰尘/烟雾/气体/雾/蒸汽/喷雾
P305+P351+P338: 如进入眼睛:用水小心冲洗几分钟。如戴隐形眼镜并可方便地取出,取出隐形眼镜。继续冲洗。
P280: 戴防护手套/穿防护服/戴防护眼罩/戴防护面具。
P302+P352: 如皮肤沾染:用水充分清洗。
P321: 特殊处理(请参阅此标签上的...)。
P405: 密闭存放
P501: 将内容物/容器处理到。。。
P264: 处理后要彻底洗手。
P271: 仅在室外或通风良好的地方使用。
P270: 使用本产品时,请勿进食、饮水或吸烟。
P304+P340: 如误吸入:将人转移到空气新鲜处,保持呼吸舒适体位。
P403+P233: 存放在通风良好的地方。保持容器密闭。
P362+P364: 脱掉沾污的衣服,清洗后方可重新使用。
P330: 漱口
P203: 使用前,获取、阅读并遵守所有安全说明。
P264+P265: 处理后彻底洗手[和…]。不要触摸眼睛。
P301+P317: 如果被吞咽:请寻求医疗帮助。
P318: 如果暴露或担心,请就医。
P317: 寻求紧急医疗救助。
P337+P317: 如果眼睛刺激持续:寻求医疗帮助。
P332+P317: 如果出现皮肤刺激:请寻求医疗帮助。
P319: 如果你感到不适,请寻求医疗帮助。
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| 批号(Lot Number) | 证书类型 | 货号 |
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| 分析证书 | I1524601 |
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