In the first article in this series, we talked about how origins of replication ori control plasmid replication and copy number. The puc19 plasmid 2,686 bp confers ampicillin resistance and complement defects in. 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. I want to do cotransfection of these plasmids in different ratios of molarity by keeping the total dna amount same 1. Since plasmid replicon type determines inc group, the terms inc and rep type were interchangeably used to describe plasmid types. Cole1type plasmids, such as pbr322 and puc, replicate in. For a transformation to be successful there are three conditions that are required, the first one is a host is needed and that is so that the dna can be inserted into something the second if a way of delivering. A desktop resource, is to curate a onestop reference guide for plasmids. For example, a highcopy puc plasmid may replicate at medium or low copy numbers when ligated to very large dna inserts, resulting in lower plasmid yields than expected.
The boiling method for isolating plasmids by holmes and quigley 1981 is presented here. How many puc18 plasmid dna molecules will be there in 10. The aim of the practical is to study the transfer of plasmid puc18 into escherichia. Naturallyoccurring plasmids are viruses of bacteria. Pdf minimal plasmids play an essential role in many intermediate steps in molecular biology.
Abstract the objective of the experiment was to engineer a puc18 plasmid so. 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 bfuai 1 433 bpmi 1 1784 bsai 1. Plasmid transformation using puc18 on e coli using heat shock method published on mar 23, 2011 determination of optimum temperature for plasmid transformation on e coli using puc 18. How many puc18 plasmid dna molecules will be there in 10 ng. In the following experiment plasmids puc18 and pkan are used to provide the genes to be transformed into bacteria. The designation puc is derived from the classical p prefix denoting plasmid and the abbreviation for. Bacterial plasmids plasmids are defined as double stranded, extrachromosomal genetic elements that replicate independently of the host cell chromosome and are stably inherited.
These changes are described in the full plasmid details pdf. Please complete this cue sheet if your production is scheduled for radio or tv broadcast, or for theatrical release. Transformation of bacteria with different plasmids objectives. The puc family 2,3 of plasmids have been extensively used as a. New vectors derives from puc 18 for clonig and thermal. Pcr amplification of the green fluorescence protein gene. Plasmid transformation using puc18 on e coli using heat shock. The cut sites for some restriction enzyme are indicated on the plasmid. Pdf engineering a minimal cloning vector from a puc18 plasmid.
Engineering a minimal cloning vector from a puc18 plasmid. Nov 26, 2014 today i would like to introduce you to puc18, a plasmid most noted for its high copy number. Vectors puc18 and puc19 are small highcopy number plasmids that are widely used for cloning and manipulation of dna fragments. Here is a graphical genealogy cro plasmid maps pdf of cro. An integrative laboratory to differentiate genetic. The pmb1 replicon rep responsible for the replication of plasmid source plasmid pbr322. Jan 14, 2015 as mentioned above, the plasmid insert can also influence copy number. The modified plasmid will have dna inserted into one of its genes.
One important feature of these plasmids is the presence of a. Why did i get genomic dna instead of plasmid dna when using. A rule of thumb to avoid bacterial genomic dna contamination during plasmid purification is to avoid vortexing during the bacterial lysis step, at the beginning of the purification protocol. In this experiment you will use the plasmid puc18 figure 1 as a cloning vector in order to amplify segments of the genome of the bacteriophage lambda. 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. The multiple cloning site mcs is within the bgalactosidase gene. Expression of target dna is enabled by the presence of a. 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. This work is licensed under the attributionnoncommercial. They can for example be used to assemble building blocks in synthetic. Another plasmid used as a vector to clone dna is called puc18 plasmid. One important feature of these plasmids is the presence of a multiple cloning site mcs within the coding region of the lacz. A versatile cloning plasmid for the expression of genes in mammalian cells.
Vectors puc18 and puc19 are small highcopy number plasmids. A plasmid is a circular dsdna molecule a few hundred or thousand base pairs in circumference. Due to a small size puc18 enables successful cloning of large dna. Vector database is a digital collection of vector backbones assembled from publications and commercially available sources.
Structural biochemistrydna recombinant techniquesplasmid. One type of the plasmid will be normal puc18 see figure 1a. This is because plasmid replication is a metabolic burden for the host cell, and if the burden becomes. The dna insert is in the middle of the lac z gene see figure 1b. No writing assistance was utilized in the production of this manuscript. The li bacterial cell was used in order to observe how its dna was able to change and develop immunity towards. This plasmid contains a gene that makes the host cell ampicillin resistant.
Minimal plasmids play an essential role in many intermediate steps in molecular biology. In the following experiment, escherichia coli were transformed using six separate agar plates. Isolation of plasmid dna from bacteria sciencedirect. Unique plasmids generated via puc replicon mutagenesis in an. A cue sheet is the sheet that radio presenters and producers use when theyre. In this first section, you will be inserting 2 types of puc18 into e. In addition, smaller plasmids generally replicate more efficiently in bacteria and produce larger numbers of plasmids per cell. Abstractthe objective of the experiment was to engineer a puc18 plasmid so that it contained a kanomycin resistance gene in its multiple cloning site and to transform it into cells. The concept was to determine cell transformation and at the same time determine. The yield of plasmid puc18 was used as transforming dna for our experiments. Psfcmvpuc18 cmv puc18 mcs plasmid plasmid vector for. Transformation occurs when plasmid dna is uptake into the bacterial cell. Initially, inc typing relied on introduction of a plasmid into a strain carrying another plasmid and determining whether both plasmids were stably maintained in the progeny. The puc18 plasmid confers ampicillin resistance and complement defects in.
Chitin nanowhiskers mediate transformation of escherichia coli by. This is a free resource for the scientific community that is compiled by addgene. The small size of this plasmid makes it less susceptible to physical damage during handling. The plasmid pgke75cata8t, which comprises puc18 and the cata8t gene encoding thermostable chloramphenicol acetyl.
Plasmid based transformations of bacterial cells are a very unique tool in current molecular biology studies. Principle foreign dna inserted at the mcs interrupts the. The puc18 and puc19 plasmids enable successful cloning of large dna fragments larger than those cloned with a m mp18 rf phage vector. Overview of primers used in the iterative shrinking of puc18 into picoz.
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. In the first article in this series, we talked about how origins of replication ori control plasmid. The kanomycin resistance gene was obtained from a pkan plasmid. Why did i get genomic dna instead of plasmid dna when. The application of these two distinct plasmids lux and puc18 in the same li strain was the focus of this work. How to manipulate plasmid copy number bitesize bio. Puc18 plasmid engineering assignment research writer. Due to a small size puc18 enables successful cloning of large dna fragments. This is not an example of the work produced by our essay writing service. A colloidal solution of chitin nanowhiskers containing puc18 plasmid dna and cells of escherichia coli was placed on an agar hydrogel and stimulated by. The plasmid puc18 and escherichia coli strains jm109 and er2925 were obtained from new england.
Radio cue sheet pdf recognise which format to include in a radio bulletin. For a transformation to be successful there are three conditions that are required, the first one is a host is needed and that is so that the dna can be inserted into something the second if a way of delivering the dna into the host which is where the vectors come in which are the two plasmids that are used lux and puc18 and the third condition. Plasmid transformation using puc18 on e coli using heat. Plasmid puc18 is a small plasmid 2686 bp that contains an ampicillinresistance gene. Transformation lab the insertion of plasmid lux and a. The vector length is 2686 bp and is isolated from e. Mossing lab dna sequences university of mississippi. According to my view, pure form of plasmid prep specifically in terms of puc18 meas all the three bands. Transformation of escherichia coli with dna from saccharomyces. These cloning vectors contain a multiple cloning site at the lacz region that enables recombinant plamids to be verified via bluewhite colony screening using agar plates containing iptg and xgal.
It also contains a gene that allows it produce beta. Engineering a minimal cloning vector from a puc18 plasmid backbone with. Mar 23, 2011 plasmid transformation using puc18 on e coli using heat shock method published on mar 23, 2011 determination of optimum temperature for plasmid transformation on e coli using puc 18. This resource is designed to educate all levels of scientists and plasmid lovers. This plasmid also contains part of the lac z gene that encode for the first 146 amino acids of. The other plasmid will be a modified form of puc18.
As many as 500 copies of this plasmid may be present in a single e. The plasmids are different in mobility and hence in size. 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. Pdf minimal plasmids play an essential role in many intermediate steps in. Heterologous expression, escherichia coli, molecular cloning, induced expression. Engineering a minimal 1185 bp cloning vector from a puc18.
Plasmid lux and puc18 in gene expression free essays. The enzyme produces a blue pigment when exposed to a specific substrate analog. The decrease in the size of the plasmid was an essential step. The desired plasmid was constructed by digesting puc18 and pkan with the same restriction enzymes,bamhi and hindiii and religating the products to. The molecule is a doublestranded circular dna 2686 base pairs in length. The purpose of this protocol is the isolation of plasmid dna from bacteria. The molecule is a small doublestranded circle, 2686 base pairs in length, and has a high copy number. It also contains a gene that allows it produce betagalactosidase, which is an enzyme degrades certain sugars. 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. Today i would like to introduce you to puc18, a plasmid most noted for its high copy number.
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