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%. 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 plasmid pgke75cata8t, which comprises puc18 and the cata8t gene encoding thermostable chloramphenicol acetyl. This system is based on a yeast strain that carries multiple integrated copies of a pucderived plasmid. Vector database is a digital collection of vector backbones assembled from publications and commercially available sources. Partitioning is strictly controlled for low copy, but loose for high copy 8. Mossing lab dna sequences university of mississippi.
Plasmid puc18 was gamma irradiated under the same conditions used for figures figures1 1 and and2. In the first article in this series, we talked about how origins of replication ori control plasmid replication and copy number. 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. A plasmid is a small, extrachromosomal dna molecule within a cell that is physically separated from chromosomal dna and can replicate independently.
Transformation lab the insertion of plasmid lux and a. Transformation of escherichia coli with dna from saccharomyces. Pdf engineering a minimal cloning vector from a puc18. 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. Here is a graphical genealogy cro plasmid maps pdf of cro. 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. It contains identical multiple cloning site mcs as puc19 vector except that it is arranged in opposite. Due to a small size puc18 enables successful cloning of large dna. 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.
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. Engineering a minimal cloning vector from a puc18 plasmid. A gel was prepared with accordance to steps in the lab manual. 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. Use text editor or plasmid mapping software to view sequence. It contains identical multiple cloning site mcs as puc19 vector except that it is arranged in opposite orientation. Dna sequences and maps tool the nucleotide sequence files available below are those used to produce the plasmid vector, viral and bacteriophage maps contained in new england biolabs catalog as well as the tables containing the locations of sites. Why did i get genomic dna instead of plasmid dna when using the plasmid dna isolation protocol. The resulting picoz plasmid is 484 bp smaller than the minimal pucmu parental plasmid. A desktop resource 1st edition 2 p a g e plasmids 101.
Pdf 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. How can i be notified when a plasmid from a specific lab or paper is available. Characterization of condensed plasmid dna models for studying. This is a more convenient and much faster procedure as compared to those used in other studies.
Naturallyoccurring plasmids are viruses of bacteria. Isolation of plasmid dna from bacteria sciencedirect. The multiple cloning site mcs is within the bgalactosidase gene. The insertion of plasmid lux and a control plasmid puc18 into escherichia coli via transformation using. Copy number is regulated by initiation of plasmid replication 10.
The puc18 plasmid confers ampicillin resistance and complement defects in. Thermo scientific puc18 vector is a small, high copy number, e. Introduction to addgenes resource any newcomer who joins a molecular biology lab will undoubtedly be asked to design. The molecule is a small doublestranded circle, 2686 base pairs in length, and has a high copy number. Amplification and cloning of taq dna polymerase gene from. How many puc18 plasmid dna molecules will be there in 10 ngul conc. The puc19 plasmid 2,686 bp confers ampicillin resistance and complement defects in. It contains identical multiple cloning site mcs as puc19. The aim of the practical is to study the transfer of plasmid puc18 into escherichia. View lab report transformation lab from bsc 1011l at florida international university. The vector length is 2686 bp and is isolated from e. Pdf engineering a minimal cloning vector from a puc18 plasmid. The designation puc is derived from the classical p prefix denoting plasmid and the abbreviation for. Plasmid replication requires host cell functions 9.
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. Today i would like to introduce you to puc18, a plasmid most noted for its high copy number. Abstract the objective of the experiment was to engineer a puc18 plasmid. Dna amplification using taq dna polymerase is one of the most widely used techniques in molecular biology and biotechnology. How many puc18 plasmid dna molecules will be there in 10 ng. Partitioning is strictly controlled for low copy, but loose for. Plasmid replication is a rigorously controlled process in part because plasmid overreplication would tax the metabolic capacity of the host cell and put the plasmidbearing cell at a disadvantage compared to. The boiling method for isolating plasmids by holmes and quigley 1981 is presented here. The vector length is 2,710 bp and is isolated from e. How much amount of recombinant plasmid dna is required for dh5 alfa competent. The bacterium of choice is a modified escherichia coli strain which allows it uptake of the plasmid puc18. Unique plasmids generated via puc replicon mutagenesis in an. Since plasmid replicon type determines inc group, the terms inc and rep type were interchangeably used to describe plasmid types.
Other reports are also available regarding the cloning of this gene 6, 9. Use with snapgene software or the free viewer to visualize additional data and align other sequences. The bacterial sequences are maintained in the yeast. 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. The aim of this study was to amplify the gene of this enzyme from a. One important feature of these plasmids is the presence of a. Isolation, cloning, and translation of plasmid dna uk essays. Why did i get genomic dna instead of plasmid dna when using. Plasmid transformation using puc18 on e coli using. New vectors derives from puc 18 for clonig and thermal.
For each radiation dose, the fraction of the plasmid in the supercoiled form was quantified by using agarose gel electrophoresis. The purpose of this protocol is the isolation of plasmid dna from bacteria. However, our study is the first to clone the taq dna polymerase gene using ta cloning method. 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. This is a free resource for the scientific community that is compiled by addgene. Plasmid dnas replicate independently of the bacterial chromosome, and many plasmids can also be. The desired plasmid was constructed by digesting puc18 and pkan with the same restriction enzymes,bamhi and hindiii and. Highlights purified by chromatography using proprietary patented technology more than. The kanomycin resistance gene was obtained from a pkan plasmid. Pdf amplification and cloning of taq dna polymerase gene.
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. Purified puc18 and pkan plasmid samples were obtained. In the first article in this series, we talked about how origins of replication ori control plasmid. Dna sequences and maps tool the nucleotide sequence files available below are those used to produce the plasmid vector, viral and bacteriophage maps contained in new england biolabs catalog as well as. 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. Plasmid sequence and snapgene enhanced annotations. One important feature of these plasmids is the presence of a multiple cloning site mcs within the coding region of the lacz. Restriction digest of puc18 dna flashcards quizlet. 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. For clarity only three different concentrations of the pentaarginine ligand are shown. Introduction to addgenes resource any newcomer who joins a molecular biology lab will undoubtedly be asked to design, modify, or construct a plasmid. The created plasmid was then transformed into li strains dh5. The molecule is a doublestranded circular dna 2686 base pairs in length.