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pBridge酵母三杂交类质粒
基本信息
启动子: ADH1,TRP1 ,MET17
复制子: 2μ ori,ori
质粒分类: 酵母系列,酵母三杂交载体
质粒大小: 6526bp
原核抗性: Amp
筛选标记: TRP1
克隆菌株: DH5α
培养条件: 37℃,有氧 LB
表达宿主: 酵母细胞
5'测序引物: M13R:CAGGAAACAGCTATGACC
3'测序引物: 根据序列设计引物
质粒简介
pBridgeTM expresses two proteins: a DNA-binding domain fusion, and an additional protein (1–3). pBridge thus allows establishment of three-hybrid systems when used in combination with an activation domain fusion vector and yeast strains from any of CLONTECH's GAL4-based two-hybrid systems (#K1604-1, #K1605-1, #K1612-1). This vector generates a hybrid protein that contains the sequences for the GAL4 DNA-binding domain (DNA-BD; a.a. 1–147) and the sequence cloned into MCS I. The fusion protein is expressed in yeast host cells from the constitutive ADH1 promoter; transcription is terminated at the ADH1 transcription termination signal. The hybrid protein is targeted to the yeast nucleus by nuclear localization sequences (NLS) that are an intrinsic part of the GAL4 DNA-BD (3). An additional gene of interest can be cloned into MCS II which is located downstream of an HA epitope and a second NLS. The resulting fusion protein is conditionally expressed from the MET25 promoter in response to methionine levels in the medium; i.e., it is repressed in the presence of 1 mM methionine and expressed in the absence of methionine (1).
pBridge is a shuttle vector that replicates autonomously in both E. coli and S. cerevisiae. It carries the bla gene (for ampicillin resistance in E. coli) and the TRP1 nutritional marker that allow yeast auxotrophs carrying pBridge to grow on limiting synthetic medium lacking tryptophan. Note: Yeast strain Y187 is a methionine auxotroph; therefore, haploid Y187 harboring pBridge cannot be grown on medium lacking methionine.
质粒图谱
扩增流程
收到产品后,请先根据产品管壁标签来判断产品形式,并在扩增前准确查找该质粒的抗性、感受态和培养温度。
1、质粒干粉(请务必转化挑单克隆重提后使用)
①收到质粒干粉后请先5000rpm离心1min,再加入20μl 无菌水溶解质粒;
②取2μl质粒加至100μl 感受态中,冰浴30min后,42℃热激60s,不要搅动,再冰浴2min;
(从第二步开始均要在超净工作台中无菌操作)
③加入500μl无抗的LB液体培养基,180rpm震荡培养45min;
④取10μl、100μl复苏液,并分别涂布至目标质粒抗性的LB平板上;
(可使用本平台的平板涂布专用玻璃珠进行涂布,可以比传统涂布方法获得更多转化子)
⑤将平板正向培养1h,再倒置37度培养16h。如果要求是30度则培养20h;
(如果菌落过多,请将质粒稀释后再转化。如果无菌落,请加入10μl质粒离心全涂转化)
⑥挑取单菌落至LB液体培养基中,加入对应抗生素,220rpm震荡培养14h,根据实验需要和质粒提取试剂盒说明书提取质粒。
2、甘油菌:四区划线后挑单菌落培养,酵母菌需要先液体复苏再四区划线,再挑单菌落液体培养。
3、穿刺菌:穿刺接种,液体培养后四区划线,再挑单菌落液体培养。
4、平板:直接挑取单菌落至液体培养基中。
5、液体质粒:单独提取的液体质粒收到后可直接使用。
pBridge酵母三杂交类质粒多少钱?
钦胜生物生物分子的应用资料有吗?
钦胜生物生物分子货号QSP0396报价含票含运吗?
钦胜生物货号QSP0396货期是多久?
QCBIOpBridge酵母三杂交类质粒可以申请试用吗?
QCBIO钦胜生物生物分子售后政策是什么?
pBridge酵母三杂交类质粒有现货吗?