产品介绍
pGEM? -T Easy 载体系统（A1360）是克隆PCR 产物的方便系统。它除了具有pGEM? -T 载体系统(Cat.# A3600, A3610) 所有的优势外，在插入位点两侧分别有EcoRI 和NotI 酶切位点，便于通过选择单酶切释放插入的DNA。pGEM? -T Easy 载体系统II 除了pGEM? -T Easy载体系统I 所有成分外，还包括JM109 感受态细胞
特点 - 优点
pGEM? -T Easy 载体系统（A1360）.
灵活：pGEM?-T Easy 载体多克隆位点两侧分别有BstZI, EcoRI和NotI 酶切位点，便于通过选择单酶切释放插入的DNA。
. 快速连接：使用2X 快速连接缓冲液(2X Rapid Ligation Buffer)可以使连接反应在室温下1 小时内完成。
. 蓝/ 白筛选：载体包含有T7 和SP6 RNA 聚合酶启动子，分别位于β - 半乳糖苷酶的α - 肽编码区内多克隆位点的两侧。α - 肽的插入失活允许在指示培养基上用颜色直接筛选重组克隆。
. f1 复制起始位点：可用于制备单链DNA。
The pGEM?-T (Cat.# A3600, A3610) and pGEM?-T Easy Vector Systems (Cat.# A1360, A1380) are convenient systems for cloning PCR products. The vectors are prepared by cutting with a restriction endonuclease to leave a blunt end and adding a 3′terminal thymidine to both ends (Figures 13.1 and 13.2). These single 3′ T overhangs at the insertion site greatly improve ligation efficiency of a PCR product into the plasmid by preventing recircularization of the vector and providing a compatible overhang for PCR products with 5' A overhangs.
The high-copy-number pGEM?-T and pGEM?-T Easy Vectors contain T7 and SP6 RNA polymerase promoters flanking a multiple cloning region within the coding region for the a-peptide of ?-galactosidase. Insertional inactivation of the a-peptide allows recombinant clones to be directly identified by color screening on indicator plates containing X-Gal (Cat.# V3941) and IPTG (Cat.# V3955). Both the pGEM?-T and pGEM?-T Easy Vectors contain numerous restriction sites within the multiple cloning region. The pGEM?-T Easy Vector multiple cloning region is flanked by recognition sites for the restriction enzymes EcoRI, BstZI and NotI, thus providing three single-enzyme digestions for release of the insert. The pGEM?-T Vector cloning region is flanked by recognition sites for the enzyme BstZI. Alternatively, a double digestion may be used to release the insert from either vector.
The pGEM?-T and pGEM?-T Easy Vectors also contain the origin of replication of filamentous phage f1 for the preparation of single-stranded DNA (ssDNA). Both pGEM?-T vector systems include a 2X Rapid Ligation Buffer for ligation of PCR products, which requires only a 1-hour incubation at room temperature. The incubation period may be extended to increase the number of colonies after transformation. Generally, an overnight incubation at 4°C will produce the maximum number of transformants.
Inserts of several kilobases have been successfully cloned into the pGEM?-T and pGEM?-T Easy Vectors (D’Avino et al. 2004). However, as the insert gets larger, the ratio of vector to insert may need to be optimized further to maximize ligation efficiency (see Ligation and Transformation in the section "Vector:Insert Ratio").
One of the disadvantages of PCR cloning into a T vector is that the insert can be cloned in either direction. Analysis of recombinant vectors by PCR or restriction enzyme digestion can be used to determine not only the success of cloning but also the insert orientation. To verify the direction of the insert, amplify recombinant plasmids using one of the gene-specific PCR primers and one of the phage promoter primers that are present on the pGEM?-T Vector (Knoche and Kephart, 1999). The correct orientation is important for transcription or translation or both.
pGEM? -T Easy 载体系统（A1360）
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