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Plasmid Puc Vector Kb Long Shown Restriction Enzymes Use Clone Gene Shown Must Clone Q

This post categorized under Vector and posted on January 30th, 2020.
PUC18 Vector: Plasmid Puc Vector Kb Long Shown Restriction Enzymes Use Clone Gene Shown Must Clone Q

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Question The Plasmid PUC18 Vector (2.7 Kb Long) Is Shown Below. What Restriction Enzymes Do You Use To Clone The Gene Shown Below You Must Clone The Entire Gene Without Breaking It Apart. There Is Only One Combination Of Enzymes. It is also critical that as much of the recipient plasmid as possible be cut with both enzymes and therefore it is important that the digest goes at least 4 hours and as long as overnight. If you are going to use only one restriction enzyme or enzymes that have compatible overhangs or no overhangs after digestion you will need to use a phosphatase to prevent re-circularization of the vector. By Mabel Derek Francis Zahra Tanya and Allison.

Ideally you will find two different restriction enzymes for your subcloning. It is also possible to use a single enzyme but this will require phosphatase treatment of your recipient plasmid as well as a specifically designed test digest later to verify that the insert was cloned in the correct orientation. A schematic representation of the pBR322 plasmid one of the first plasmids to be used widely as a cloning vector. Shown on the plasmid diagram are the genes encoded (amp and tet for ampicillin and tetracycline resistance respectively) its origin of replication (ori) and various restriction sites (indicated in blue). It is not always necessary to use an IP- heavy kit as traditional cloning by restriction enzyme digestion is made even easier with standardized plasmids from Oxford Genetics. The aim at Oxford Genetics was to engineer a DNA plasmid system that could accommodate most of the functional DNA inserts that a researcher might require within a single plasmid.

pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers. The designation pUC is derived from the clvectorical p prefix (denoting plasmid) and the abbreviation for the University of California where early work on the plasmid series had been conducted.It is a circular double stranded DNA and has 2686 base pairs. 1. A circular bacterial plasmid (pBP1) has a single HindIII restriction-enzyme site in the middle of a tetracycline-resistance gene (tet R).Fruit fly genomic DNA is digested with HindIII and a library is made in pBP1. Probing reveals that clone 15 contains a specific Drosophila gene of interest. Clone 15 is studied by restriction vectorysis with HindIII and another restriction enzyme EcoRV. Most plasmid vectors contain little more than the essential nucleotide sequences required for their use in DNA cloning areplication origin a drug-resistance gene and a region in which exogenous The essence of cell chemistry is to isolate a particular cellular component and then vectoryze its chemical structure and activity. In the case of DNA this is feasible for relatively short molecules such as the genomes of small viruses. But genomes of even the simplest cells are much too large to directly vectoryze in detail at the molecular level.
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