They are most commonly found as small circular, doublestranded dna molecules in bacteria. The cloning vector puc18 was cleaved with the help of smai restriction enzyme. Overview of primers used in the iterative shrinking of puc18 into picoz. Plasmid lux and puc18 in gene expression free essays. This is a free resource for the scientific community that is compiled by addgene this page is informational only this vector is not available from addgene please contact the manufacturer for further details. Today i would like to introduce you to puc18, a plasmid most noted for its high copy number. Plasmid transformation using puc18 on e coli using heat. Welcome to vector database vector database is a digital collection of vector backbones assembled from publications and commercially available sources. Cmv puc18 mcs plasmid psfcmvpuc18sequence txtthis plasmid is. It has been constructed using the ampicillin resistance gene and the pmb1 origin of.
These mutations were first noted in the highcopy plasmid, puc18. Subsequently added 15l of nuclease free water followed by 2. The pmb1 replicon rep responsible for the replication of plasmid source plasmid pbr322. It can be achieved a spontaneously as in replicaplated colonies. If desired, the plasmid can be exported as a genbank file. May 24, 2019 the resulting picoz plasmid is 484 bp smaller than the minimal pucmu parental plasmid. The artificial plasmid puc18 has been genetically engineered to include 1 a gene for antibiotic resistance to ampicillin amp r, and 2 a gene and its promoter for the enzyme betagalactosidase lacz.
Most cloning vectors have unique restriction sites. Now we are going to learn about genetic alterations in plasmids that affect copy number. Multiplicity of dna singlestrand breaks produced in puc18 exposed to the direct effects of ionizing radiation. Insertion of foreign dna into plasmids from grampositive bacteria. The process of obtaining plasmidfree isolates is termed curing. The molecule is a small doublestranded circle, 2686 base pairs in length, and has a high copy number. In the following experiment plasmids puc18 and pkan are used to provide the genes to be transformed into bacteria. A versatile cloning plasmid for the expression of genes in mammalian cells. Principle foreign dna inserted at the mcs interrupts the. To see this sequence with restriction sites, features, and translations, please download snapgene or the free snapgene viewer. Multiplicity of dna singlestrand breaks produced in puc18. People developing this backbone further are naturally free to explore and adapt it.
The aim of the practical is to study the transfer of plasmid puc18 into escherichia. Pdf minimal plasmids play an essential role in many intermediate steps in. The cut sites for some restriction enzyme are indicated on the plasmid. The vector length is 2686 bp and is isolated from e. With these four sequential steps of elimination, we have thus been able to reduce the 2686bp puc18 plasmid to the 1185bp picoz plasmid, which contains more useable cloning sites, a total size reduction of 56%. Plasmid elements unique restriction sites for cloning the plasmid needs too be linearized. Radiation chemical yields in nmolj for free base release in puc18 plasmid dna films and calculation of the multiplicity of singlestrand breaks we found that the yield of total free base release, g fbr, increased by 60% as the hydration was increased from a.
Restriction enzyme cut sites can also be specified or automatically identified. Engineering a minimal cloning vector from a puc18 plasmid backbone with an extended multiple cloning site article pdf available in biotechniques 666 may 2019 with 219 reads. Sometimes, a plasmid is said to be lost when the progeny cells dont receive the plasmid. This material is available to academics and nonprofits only.
The puc family 2,3 of plasmids have been extensively used as a. Pdf engineering a minimal cloning vector from a puc18. In the first article in this series, we talked about how origins of replication ori control plasmid replication and copy number. The concept was to determine cell transformation and at the same time determine. Although the newcomer likely knows that a plasmid is a small circular piece of dna found in bacterial cells, she may need some extra guidance to understand the specific components that make up a plasmid and why each is important.
Plasmid based transformations of bacterial cells are a very unique tool in current molecular biology studies. The plasmids are different in mobility and hence in size. New vectors derives from puc 18 for clonig and thermal. The high copy number of puc plasmids is a result of the lack of the rop gene and a single point mutation in the replicon rep of pmb1. Vector database is a digital collection of vector backbones assembled from publications and commercially available sources. The designation puc is derived from the classical p prefix denoting plasmid and the abbreviation for the university of california, where early work on the plasmid series had been conducted.
The bacterium of choice is a modified escherichia coli strain which allows it uptake of the plasmid puc18. This plasmid contains the multiple cloning site mcs from puc18 however it has been modified slightly to accommodate some restriction sites in our vector system. The application of these two distinct plasmids lux and puc18 in the same li strain was the focus of this work. A plasmid is a small, extrachromosomal dna molecule within a cell that is physically separated from chromosomal dna and can replicate independently. Plasmid is a more descriptive as it is a type of vector. Please acknowledge the principal investigator, cite the article. The latter one, pucp18, has a broad host range origin that was cloned into the puc18 plasmid, so it will replicate in many gram negative bacteria. Using a pipette, 70 ul of earlier prepared competent cells were transferred into each of the tubes containing the plasmids. We are happy to participate in genome compilers free, w. Plasmid uptake by competent cells were subsequently demonstrated by using 3 ul each of test plasmid lux and control plasmid puc18 in tubes labeled accordingly and kept cold in ice.
Naturallyoccurring plasmids are viruses of bacteria. The li bacterial cell was used in order to observe how its dna was able to change and develop immunity towards. Please practice handwashing and social distancing, and check out our resources for adapting to these times. People developing this backbone further are naturally free to explore and. Vectors puc18 and puc19 are small highcopy number plasmids that are widely used for cloning and manipulation of dna fragments. The yield of plasmid puc18 was used as transforming dna for our experiments. Psfcmvpuc18 cmv puc18 mcs plasmid plasmid vector for molecular cloning.
One important feature of these plasmids is the presence of a multiple cloning site mcs within the coding region of the lacz. Aatii 1 2617 acc65i 1 408 acci 1 429 afliii 1 806 ahdi 1 1694 alwni 1 1217 apoi 1 396 avai 1 412 bamhi 1 417 banii 1 402 bcgi 1 2215. Similarly to how a cheetah is both a mammal and an animal. Lysed cell zone contains free phage particles and proteins. Plasmids pub110, pc194, pe194, and pt181 are commonly used as cloning. If the plasmid contains more than one site for a given restriction enzyme, this results in fragmentation of the plasmid why does this matter.
Engineering a minimal cloning vector from a puc18 plasmid. To learn about this, we focused on the pbr22 ori and the role of rop protein in controlling copy number within pbr22 and other members of the cole1 family. The transition of plasmid dna from a supercoiled to an open circle conformation, as detected by gel electrophoresis, affords an extraordinarily sensitive method for detecting singlestrand breaks. Pdf engineering a minimal cloning vector from a puc18 plasmid. Nov 26, 2014 to learn about this, we focused on the pbr22 ori and the role of rop protein in controlling copy number within pbr22 and other members of the cole1 family.
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