Puc18 plasmid pdf download

Transformation lab the insertion of plasmid lux and a. The vector length is 2686 bp and is isolated from e. Vectors puc18 and puc19 are small highcopy number plasmids that are widely used for cloning and manipulation of dna fragments. Due to a small size puc18 enables successful cloning of large dna fragments. The resulting picoz plasmid is 484 bp smaller than the minimal pucmu parental plasmid. Photochemical methods to assay dna photocleavage using. The kanomycin resistance gene was obtained from a pkan plasmid. May 24, 2019 the resulting picoz plasmid is 484 bp smaller than the minimal pucmu parental plasmid. Plasmid pbr322 was specifically constructed as a cloning vector, and is the basis of many of the more modem vectors see section 11. You will estimate the quantity of dna present in the puc18 dna prep by gel band brightness.

They can for example be used to assemble building blocks in synthetic biology or be used as intermediate cloning plasmids that are ideal for pcrbased mutagenesis methods. 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. Pdf minimal plasmids play an essential role in many intermediate steps in molecular biology. The pmb1 replicon rep responsible for the replication of plasmid source plasmid pbr322. 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%. Use with snapgene software or the free viewer to visualize additional data and align other sequences. It contains identical multiple cloning site mcs as puc19 vector except that it is arranged in opposite orientation. Apr 04, 2015 the cloning site is a site in the plasmid where the dna is inserted. The li bacterial cell was used in order to observe how its dna was able to change and develop immunity towards ampicillin.

Engineering a minimal cloning vector from a puc18 plasmid. Zebrafish dna sequence from clone busm163m10 in linkage group 1 contains three novel genes for mhc class i antigens, a novel gene similar to human c21orf18 chromosome 21 open reading frame 18 and a novel gene similar to human cryzl1 crystallin, zeta quinone reductaselike 1. By selecting the box below, i am agreeing to allow addgene, via its ebook service provider, hubspot, to store my email address indefinitely or until i opt out of receiving updates to the plasmids 101 ebook so that i can receive the plasmids 101 ebook and any updates to this ebook at the email address i provided above. A desktop resource 1st edition 2 p a g e plasmids 101. The map, notes, and annotations on this page and in the sequencemap file are ed material. Multiplicity of dna singlestrand breaks produced in puc18. The aim of the practical is to study the transfer of plasmid puc18 into escherichia. Transformation was carried out as described in kushner et al. Pdf puc18cpg is an effective adjuvant for a duck tembusu. Zebrafish dna sequence from clone busm163m10 in linkage group 1 contains three novel genes for mhc class i antigens, a novel gene similar to human c21orf18 chromosome 21 open reading frame 18 and a novel gene similar to human cryzl1 crystallin, zeta quinone reductaselike 1, complete sequence. It can be downloaded from the detaisl view of the puc18puc19 product description.

It contains identical multiple cloning site mcs as puc19. New vectors derives from puc 18 for clonig and thermal. Organisms whose genes have been artificially altered for a desired affect is often called genetically modified organism gmo. Although the newcomer likely knows that a plasmid is a small circular piece of dna found in bacterial cells, she may. Plasmid transformation using puc18 on e coli using. Plasmids are first type of cloning vector developed. Engineering a minimal cloning vector from a puc18 plasmid backbone with an extended multiple. Recombinant dna technology rdna is technology that is used to cut a known dna. 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.

Introduction to addgenes resource any newcomer who joins a molecular biology lab will undoubtedly be asked to design, modify, or construct a plasmid. 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. Hybrid plasmid composed of the replicon and kmr gene of publlo. The insertion of plasmid lux and a control plasmid puc18 into escherichia coli via transformation using. Plasmid lux and puc18 in gene expression free essays. Minimal plasmids have many uses and can either be generated synthetically 1 or by iterative deletions in an existing plasmid. Us6127174a plasmid derived from gluconobacter bacteria. 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. Multiplicity of dna singlestrand breaks produced in puc18 exposed to the direct effects of ionizing radiation. 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.

Since plasmid replicon type determines inc group, the terms inc and rep type were interchangeably used to describe plasmid types. The cloning site is a site in the plasmid where the dna is inserted. The plasmids are different in mobility and hence in size. 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. A sister vector puc18 contains an inverted version of the multiple cloning sites mcs. 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. Principle foreign dna inserted at the mcs interrupts the.

The puc18puc19 sequence is stored as a pdf file on the genaxxon webpage. The multiple cloning site mcs is within the bgalactosidase gene. I did go through the fermentas website, and found the restriction map of puc18 i have attached it for clarity but couldnt decipher it. The puc18puc19 sequence is stored as a pdffile on the genaxxon webpage it can be downloaded from the. Use text editor or plasmid mapping software to view sequence. The resolution of the dna bands using gel electrophoresis makes circular plasmid dna like puc18 ideal as a qualitative probe of photochemistry that causes cleavage of the dna backbone.

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. 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. Pdf engineering a minimal cloning vector from a puc18. 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. The puc18 plasmid confers ampicillin resistance and complement defects in. Naturallyoccurring plasmids are viruses of bacteria. Mobilizable narrowhostrange plasmids were constructed from puc18 and puc19 by addition of a segment of.

Insertion of foreign dna into plasmids from grampositive bacteria. Engineering a minimal cloning vector from a puc18 plasmid backbone with an extended multiple cloning site. In the first article in this series, we talked about how origins of replication ori control plasmid replication and copy number. It is a commonly used cloning vector of size 2686 bp with an ampicillin resistance gene. Engineered plasmids can be used to clone genetic material of up to 10,000 base pairs. Plasmidology the inclusion of polylinkers into plasmid vectors polylinker is a tandem array of restriction endonuclease sites in a very short expanse of dna for example, puc18s polylinker sites for res region spans the equivalent of 20 amino acids or 60 nucleotides. The molecule is a small doublestranded circle, 2686 base pairs in length, and has a high copy number.

Today i would like to introduce you to puc18, a plasmid most noted for its high copy number. View lab report transformation lab from bsc 1011l at florida international university. Genetic engineering recombinant dna technology genetic engineering is a broad term referring to manipulation of an organisms nucleic acid. The created plasmid was then transformed into li strains dh5. I just was curious about the size of the fragments resulting from the digestion of the vector puc18 with hindiii first and then ecori. Unique plasmids generated via puc replicon mutagenesis in an.

Geneitm plasmid preparation teaching kit manual cat no. The puc family 2,3 of plasmids have been extensively used as a backbone for various cloning and expression vectors 4. Construction of mobilizable vectors derived from plasmids rp4. The puc19 plasmid 2,686 bp confers ampicillin resistance and complement defects in. The cut sites for some restriction enzyme are indicated on the plasmid. The designation puc is derived from the classical p prefix denoting plasmid.

Plasmids were prepared by the boiling protocol from microscale cultures of dh5. One such benefit is the ability to produce large quantities of biological materials that. A shuttle vector autonomously replicable in bacteria belonging to the genera gluconobacter and escherichia is constructed from an endogenous plasmid of gluconobacter oxydans if03171 strain, which has a size of about 5. The puc family 2,3 of plasmids have been extensively used as backbone for various cloning and expression vectors 4. Bacterial plasmids can not accept dna strands larger than 5000 base pairs, hence they are restricted to cloning dna. The molecule is a doublestranded circular dna 2686 base pairs in length.

We biology lab cloning paper plasmid answer pdf on this page you can read or download lab cloning paper plasmid worksheet ms foglia ap biology in pdf format. Plasmid are not necessary for survival but may confer some advantage to the. Another famous plasmid that was specifically constructed for use as a cloning vector is pacyc184 chang and cohen, 1978. Compare your linear plasmid band in the hindiii digest lane to the. Pdf engineering a minimal cloning vector from a puc18 plasmid. Highlights purified by chromatography using proprietary patented technology more than. Kt06 106216 kt06a 106217 kt06b 106218 revision no 01280906. New vectors derives from puc 18 for clonig and thermalinduced expression in escherichia coli. Minimal plasmids play an essential role in many intermediate steps in molecular biology. Nov 26, 2014 today i would like to introduce you to puc18, a plasmid most noted for its high copy number. A small backbone also opens up for additional unique restriction enzyme cloning sites.

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. Plasmid sequence and snapgene enhanced annotations. Welcome to vector database vector database is a digital collection of vector backbones assembled from publications and commercially available sources. The yield of plasmid puc18 was used as transforming dna for our experiments.