Modified cloning vector and application thereof

Abstract

The invention relates to a modified cloning vector and an application thereof. The modified cloning vector is an annular cloning vector, and is obtained by replacing multiple cloning sites of a pUC57 vector by employing three flush end cloning sites of EcoRV, SmaI and StuI and removing an NdeI enzyme digestion site at the LacZ downstream in the pUC57 vector, and the modified cloning vector only comprises three cloning sites of EcoRV, SmaI and StuI. The vector can completely replace any one cloning vector and is convenient to use and is wide in application.

Description

technical field [0001] The invention relates to a cloning carrier, in particular to a carrier convenient for gene cloning work. Background technique [0002] Currently, there are two types of cloning vectors commonly used in the field of molecular biology; one is a circular cloning vector with a large number of multiple cloning sites, and the other is a linear T vector without multiple cloning sites. But they all have certain usage restrictions. [0003] A circular cloning vector with multiple cloning sites, taking pUC57 as an example, its multiple cloning site contains EcoRI, HindIII and other commonly used enzyme cutting sites, but after all, it cannot contain all enzyme cutting sites, such as the commonly used NotI , XhoI, etc., and often the inserted target gene needs to use these unrestricted restriction sites, so double digestion cloning is subject to certain restrictions, unless additional restriction sites are added at both ends of the target gene. If blunt-end clo...

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Problems solved by technology

But they all have certain usage restrictions

[0003] A circular cloning vector with multiple cloning sites, taking pUC57 as an example, its multiple cloning site contains EcoRI, HindIII and other commonly used enzyme cutting sites, but after all, it cannot contain all enzyme cutting sites, such as the commonly used NotI , XhoI, etc., and often the inserted target gene needs to use these unavailable restriction sites, so double restriction cloning is subject to certain restrictions, unless additional restriction sites are added at both ends of the target gene

If blunt-end cloning is used, sometimes the target gene has its own restriction site on the vector, which results in multiple restriction sites in the final constructed recombinant plasmid, and the uniqueness of the restriction site is affected. will have some impact on subsequent experiments.

[0004] Linear T vector cloning, taking pMD18-T simple as an example, T vector can only clone PCR products with A tail, but now the use of high-fidelity enzymes (such as PFU) is becoming more and more popular, and the products amplified with PFU are all blunt end, which brings some troubles to TA cloning

At the same time, linear carriers cannot be copied in large quantities, so technology and cost are often limited by manufacturers, and they cannot be self-sufficient

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