The purpose of this protocol is the isolation of plasmid dna from bacteria. The enzyme produces a blue pigment when exposed to a specific substrate analog. In vitro crisprcas9 system for efficient targeted dna editing. The puc18puc19 sequence is stored as a pdffile on the genaxxon. Sticky ends from different bfuai sites may not be compatible. 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. 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. Today i would like to introduce you to puc18, a plasmid most noted for its high copy number. In the first article in this series, we talked about how origins of replication ori control plasmid replication and copy number. Since plasmid replicon type determines inc group, the terms inc and rep type were interchangeably used to describe plasmid types. One important feature of these plasmids is the presence of a multiple cloning site mcs within the coding region of the lacz. Structural biochemistrydna recombinant techniquesplasmid.
To understand dna moves through agarose gel based on size and charge differentiation. 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. For example, they can be used to assemble building blocks in synthetic biology or be used as intermediate cloning plasmids that are ideal for pcrbased mutagenesis methods. The kanomycin resistance gene was obtained from a pkan plasmid. The yield of plasmid puc18 was used as transforming dna for our experiments. With these four sequential steps of elimination, we have thus been able to reduce the 2686 bp puc18 plasmid to the 1185 bp picoz plasmid which contains more useable cloning sites, a total size reduction of 56%. Methods for the study of dna photocleavage are illustrated using a mixedmetal supramolecular complex bpy 2 rudpp 2 rhcl 2cl 5. From the white paper, cut out the puc18 plasmid dna in a long strip. The plasmid puc18 plasmid can then be used to transform bacteria so that it now expresses a new gene and produces a new protein. 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. A repository of over 200,000 plasmids including protein structure initiative protein expression plasmids and vectors, over 75,000 human plasmids, and whole genome collections from many organisms. 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.
Because there are polyclonal sites in the lacz gene, it is very easy to determine whether there are insertions of dna fragments in the vector through the blue and. These changes are described in the full plasmid details pdf. It is a circular double stranded dna and has 2686 base pairs. A plasmid is a circular dsdna molecule a few hundred or thousand base pairs in circumference. Plasmid sequence and snapgene enhanced annotations.
Photochemical methods to assay dna photocleavage using. The pmb1 replicon rep responsible for the replication of plasmid source plasmid. Bfuai is typically used at 50c, but is 50% active at 37c. A repository of over 200,000 plasmids including protein structure initiative protein expression plasmids and vectors, over 75,000 human plasmids, and. Highlights purified by chromatography using proprietary patented technology more than. Ms foglia ap biology answers cloning paper plasmid ms foglia ap biology lab cloning paper plasmid answers. 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 206 reads. Pdf engineering a minimal cloning vector from a 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. Isolation of plasmid dna from bacteria sciencedirect. The boiling method for isolating plasmids by holmes and quigley 1981 is presented here.
Psfcmvpuc18 cmv puc18 mcs plasmid plasmid vector for. Thermo scientific puc18 vector is a small, high copy number, e. Detailed methods for performing the photochemical experiments and analysis of the dna photoproduct are delineated. New vectors derives from puc 18 for clonig and thermal. The clustered regularly interspaced short palindromic repeat crisprcrisprassociated protein 9 cas9 system, an rnaguided nuclease for specific genome editing in vivo, has been adopted in a wide variety of organisms. The artificial plasmid puc18 has been genetically engineered to include 1 a gene for antibiotic resistance.
The puc family 2,3 of plasmids have been extensively used as backbone for various cloning and expression. 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. It can be downloaded from the detaisl view of the puc18puc19 product description. The methods use supercoiled puc18 plasmid as a dna probe and either filtered light from a xenon arc lamp source or monochromatic light from a newly designed, highintensity lightemitting diode led array. One such benefit is the ability to produce large quantities of biological materials that were previously difficult to obtain. The created plasmid was then transformed into li strains dh5. Because there are polyclonal sites in the lacz gene, it is very easy to determine whether there. Plasmid dnas replicate independently of the bacterial chromosome, and many plasmids can also be. Efficient cleavage requires at least two copies of the bfuai recognition sequence. It contains identical multiple cloning site mcs as puc19 vector except that it is arranged in opposite orientation. The desired plasmid was constructed by digesting puc18 and pkan with the same restriction enzymes,bamhi and hindiii and religating the products to give the engineered puc18.
Nov 26, 2014 today i would like to introduce you to puc18, a plasmid most noted for its high copy number. Efficient cleavage requires at least two copies of the bspmi recognition sequence. This method is rapid and simple and it allows for a large number of samples to. The puc family 2,3 of plasmids have been extensively used as. Principle foreign dna inserted at the mcs interrupts the. Some commercial stocks of puc18 were reported to be contaminated by a mutant plasmid with a mutation in the second codon of the lacz gene, resulting in the diminished intensity of the blue colony color. Engineering a minimal cloning vector from a puc18 plasmid backbone with an extended multiple cloning site.
Here is a graphical genealogy cro plasmid maps pdf of cro expression plasmids from the sauer lab. The resulting picoz plasmid is 484 bp smaller than the minimal pucmu parental plasmid. By continuing to use this site, you agree to the use of cookies. Vectors puc18 and puc19 are small highcopy number plasmids that are widely used for cloning and manipulation of dna fragments. Naturallyoccurring plasmids are viruses of bacteria. The plasmids are different in mobility and hence in size. New vectors derives from puc 18 for clonig and thermalinduced expression in escherichia coli. It also contains a gene that allows it produce betagalactosidase, which is an enzyme degrades certain sugars. 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 effects of plasmid on genotype and phenotype revised 196 introduction plasmids are small circular dna molecules that often found in bacteria in addition to the large circular dna molecule of the bacterial chromosome.
Because puc18sfi is a derivative vector from puc18, both of vectors have common. Another plasmid used as a vector to clone dna is called puc18 plasmid. Use text editor or plasmid mapping software to view sequence. Since the rop protein is a negative regulator of plasmid replication within the cole1 family, it is clear that its absence will loosen the control on plasmid replication leading to increased copy number. The pmb1 replicon rep responsible for the replication of plasmid source plasmid pbr322. A sister vector puc18 contains an inverted version of the multiple cloning sites mcs. A versatile cloning plasmid for the expression of genes in mammalian cells. Use with snapgene software or the free viewer to visualize additional data and align other sequences.
Article engineering a minimal cloning vector from a puc18. The molecule is a small doublestranded circle, 2686 base pairs in length, and has a high copy number. Minimal plasmids play an essential role in many intermediate steps in molecular biology. The map, notes, and annotations on this page and in the sequencemap file are ed material.
In contrast, the in vitro application of the crisprcas9 system has rarely been reported. Pdf engineering a minimal cloning vector from a puc18 plasmid. The vector length is 2686 bp and is isolated from e. Plasmid transformation using puc18 on e coli using. The aim of the practical is to study the transfer of plasmid puc18 into escherichia. A small backbone also opens up for additional unique restriction enzyme cloning sites. Home resources plasmid files basic cloning vectors puc18. Download file pdf biology lab cloning paper plasmid answers are used by molecular biologists to insert a geneofinterest into a vector.
On this page you can read or download ms foglia ap biology lab cloning paper plasmid answers in pdf format. To characterize dna band fragments using restriction enzymes. On this page you can read or download lab cloning paper plasmid answer in pdf format. Sulfonylurea resistance as a new selectable marker for the. 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. It has been constructed using the ampicillin resistance gene and the pmb1 origin of. This plasmid contains a gene that makes the host cell ampicillin resistant. The cut sites for some restriction enzyme are indicated on the plasmid. This website uses cookies to ensure you get the best experience.
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