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查看更多产品信息 TOPO® XL PCR Cloning Kit, with One Shot® TOP10 Electrocomp™ E. coli - FAQs (K470020)
9 个常见问题解答
两个载体的骨架非常相似。主要的不同在于pCR4-TOPO载体测序引物位点距离PCR产物插入位点仅33 bp。这使得对插入序列进行测序时需要读取的载体序列能最小化，从而使pCR4-TOPO载体非常适合进行测序应用。
两个载体的骨架非常相似。主要的不同在于pCRII-TOPO 载体是一个双启动子载体，包含SP6和T7启动子用于体外转录/测序，而 pCR2.1-TOPO仅包含T7启动子用于体外转录/测序。两个载体均含有M13 正向和反向引物位点用于测序或PCR检测。
对照模板的序列是受专利保护的。
为了提高凝胶中结晶紫染色的强度，请将凝胶置于结晶紫染色液（将45 µL浓度为2 mg/mL染色剂加入到100 mL无菌水中或在0.1X TAE中加入1-10 µg/mL染色剂）中数小时，染色会加深一些。切胶时为了看得更清晰可以把凝胶放在白色背景下。
The vector backbones for both of these vectors are very similar. The main difference is that the pCR4-TOPO vector has sequencing primer sites located as close as 33 base pairs from the PCR product insertion site. This minimizes the amount of vector DNA sequence that needs to be read before reaching the sequence of the insert, making the pCR4-TOPO vector very useful for sequencing applications.
The vector backbones for both of these vectors are very similar. The main difference is that the pCRII-TOPO vector is a dual promoter vector, containing the SP6 and T7 promoters for in vitro transcription/sequencing, whereas the pCR2.1-TOPO vector contains only the T7 promoter for in vitro transcription/sequencing. Both vectors contain the M13 Forward and Reverse primer sites for sequencing or PCR screening.
The sequence of the control template is proprietary.
To increase the intensity of the crystal violet-stained gel, allow the gel to lie for a couple of hours in crystal violet stain (45 µL of 2 mg/mL stain in 100 mL sterile water or 1-10 µg/mL stain in 0.1X TAE) and it will stain a little darker. Place the gel over a white background when excising the band to improve visibility.
Typically 200-300 colonies are obtained for the TOPO kits with 100% inserts (20/20). For the Zero Blunt kit that uses standard ligase, typically 600-1000 colonies are obtained with 95-100% inserts.