Plasmid transformation using puc18 on e coli using. In addition, smaller plasmids generally replicate more efficiently in bacteria and produce larger numbers of plasmids per cell. What is the difference between puc18 and puc19 answers. Use with snapgene software or the free viewer to visualize additional data and align other sequences. The map, notes, and annotations on this page and in the sequencemap file are ed material. The yield of plasmid puc18 was used as transforming dna for our experiments. Article engineering a minimal cloning vector from a puc18. Pcr amplification of the green fluorescence protein gene for.
The puc19 plasmid 2,686 bp confers ampicillin resistance and complement defects in. Heterogeneity in surface sensing suggests a division of labor. It contains the pmb1 origin of replication from pbr322, but it lacks the rop gene and carries a point mutation in the rnaii transcript g 2975 in pbr322 to a 8 in puc19. Transformation lab the insertion of plasmid lux and a.
The pmb1 replicon rep responsible for the replication of plasmid source plasmid pbr322. Plasmid are not necessary for survival but may confer some advantage to the. 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. This is a free resource for the scientific community that is compiled by addgene. The plasmid had a 4878bp segment of bacteriophage lambda inserted into the ecor1 site, thus after digest. Disease control in shrimp aquaculture with probiotic. Psfcmvpuc18 cmv puc18 mcs plasmid plasmid vector for. The aim of the practical is to study the transfer of plasmid puc18 into escherichia.
It is a circular double stranded dna and has 2686 base pairs. 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. 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. Coli on ampicillin plates demonstrates transformation to an antibioticresistant phenotype.
The kanomycin resistance gene was obtained from a pkan plasmid. The cut sites for some restriction enzyme are indicated on the plasmid. Plasmid dnas replicate independently of the bacterial chromosome, and many plasmids can also be. The plasmids are different in mobility and hence in size. The puc18 plasmid confers ampicillin resistance and complement defects in. The transformation efficiency of bl21ai cells is 1 x 108 cfu. Vectors puc18 and puc19 are small highcopy number plasmids that are widely used for cloning and manipulation of dna fragments. How to manipulate plasmid copy number bitesize bio. Plasmid sequence and snapgene enhanced annotations. 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.
Pdf engineering a minimal 1185 bp cloning vector from a puc18. Testing plasmid size and equal molar amounts on transformation frequency plasmids were obtained smit laboratory, ubc that allowed for testing the effects of increasing plasmid size on transformation frequency, and that closely resemble the construct we had tried to create above. Engineering a minimal cloning vector from a puc18 plasmid backbone with an extended multiple cloning site. Minimal plasmids have many uses and can either be generated synthetically 1 or by iterative deletions in an existing plasmid. The puc18 plasmid structure consists of the ampicillin resistance gene, a gene that.
Easypreptm plasmid miniprep 5 6 kit contents at 5,000 g for 10 min or 3,500 rpm for 20 min in a tabletop pellet by vortexing or pipetting. Although the newcomer likely knows that a plasmid is a small circular piece of dna found in bacterial cells, she may. The small size of this plasmid makes it less susceptible to physical damage during handling. Genetic engineering allows multiple sites to be inserted into the dsdna loop such as the gene and promoter for betagalactosidase, lacz.
The primers for sequencing an insert in the polykinker region of these plasmids are what most labs refer to as universal m primers. Plasmid is a more descriptive as it is a type of vector. 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. Dna purification and isolation of genomic dna from. Pdf molecular cloning and expression of the lux genes of vibrio. Naturallyoccurring plasmids are viruses of bacteria. The boiling method for isolating plasmids by holmes and quigley 1981 is presented here. Today i would like to introduce you to puc18, a plasmid most noted for its high copy number. One important feature of these plasmids is the presence of a multiple cloning site mcs within the coding region of the lacz. Using lux and puc18 plasmids in the bacterial transformation of escherichia coli for resistance against. In the basic version of laboratory 5, the observed phenotype was due to uptake of plasmid pamp, a dna molecule that is wellcharacterized.
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. They are most commonly found as small circular, doublestranded dna molecules in bacteria. Feb252005 396452 multiple cloning site ecorihindiii is there any general rules concerning the size of an insert versus the size of a plasmid. A desktop resource 1st edition 2 p a g e plasmids 101. Dna axial rotation and the merge of oppositely supercoiled. Plasmid dna from escherichia coli rri d3404 datasheet. Engineering a minimal cloning vector from a puc18 plasmid backbone with an extended multiple cloning site preprint pdf available october 2018 with 475 reads how we measure reads.
Into this region has been inserted a piece of dna called a polylinker, or multiple cloning site, which contains many unique restriction target sites useful for inserting. Another plasmid used as a vector to clone dna is called puc18 plasmid. The insertion of plasmid lux and a control plasmid puc18 into escherichia coli via transformation using. 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. What primers should i use to sequence an insert puc19, pneb193, litmus. The multiple cloning site mcs is within the bgalactosidase gene. Copy number is regulated by initiation of plasmid replication 10. Apr 16, 2014 istiqomah b1j012019 tugas terstruktur biologi molekuler universitas jenderal soedirman. Multiple antibiotic resistance plasmids allow scalable ncbi. How many puc18 plasmid dna molecules will be there in 10 ngul conc. Since plasmid replicon type determines inc group, the terms inc and rep type were interchangeably used to describe plasmid types. Thermo scientific puc18 vector is a small, high copy number, e. The created plasmid was then transformed into li strains dh5.
The bacterium of choice is a modified escherichia coli strain which allows it uptake of the plasmid puc18. The lacz gene contains a polylinker region that is 54 base pairs in length and contains different restriction sites for restriction endonucleases involved in procedures such as dna. These changes are described in the full plasmid details pdf. This method is rapid and simple and it allows for a large number of samples to be processed simultaneously up to 40 samples. Neb offers its own series of these primers, shown in the neb catalog. Pdf engineering a minimal cloning vector from a puc18. Pdf insertion of unknown genomic dna into puc 18 and. 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. Terms and conditions 20042018 harvard medical school plasmid was created and is maintained by the dfhcc dna resource core at harvard medical schooldfhcc dna resource core at harvard medical school. How can i be notified when a plasmid from a specific lab or paper is available.
This plasmid contains a gene that makes the host cell ampicillin resistant. Snapgene is a very complete, yet remarkably userfriendly application. In most instances, plasmid dna, isolated by these procedures, is of sufficient quality and quantity for cloning and pcr analysis. Expression vector for adjustable expression of exogenous genes in prokaryotes. A versatile cloning plasmid for the expression of genes in mammalian cells. Introduction to addgenes resource any newcomer who joins a molecular biology lab will undoubtedly be asked to design, modify, or construct a plasmid. New vectors derives from puc 18 for clonig and thermal. Structural biochemistrydna recombinant techniquesplasmid. This was done by isolation of the genomic dna and plasmid dna using. Our entire lab adopted it without any reservations and it has proved to be the ideal tool for easy and thorough documentation of all the constructs we are routinely generating.
Jan 14, 2015 how to manipulate plasmid copy number published january 14, 2015 so far in this series, we have looked at origins of replication, weve discussed how plasmid replication is regulated in the popular pbr22 plasmid, and weve seen how a disturbance of this regulatory mechanism has given rise to the highcopy puc18 plasmid. Taq dna polymerase or a polymerase blend recommended for pcr cloning. Dna were amplified by pcr and these, 2000, and 4000 bp fragments were cloned into puc19 vectors. The effect of increasing plasmid size on transformation. Strataclone solopack competent cells and puc18 control plasmid. How much amount of recombinant plasmid dna is required for dh5 alfa competent. The puc plasmid is a more advanced vector, whose structure allows direct visual selection of colonies containing vectors with donor dna inserts.
It contains identical multiple cloning site mcs as puc19 vector except that it is arranged in opposite orientation. Combining two or three resistance genes simultaneously on the same dna vector. Doc formation of recombinant puc18 plasmid from restriction. What primers should i use to sequence an insert puc19. The vector length is 2686 bp and is isolated from e.
One such benefit is the ability to produce large quantities of biological materials that were previously difficult to obtain. Isolation of plasmid dna from bacteria sciencedirect. Dna fragments into puc19 vector to study the ligation. Principle foreign dna inserted at the mcs interrupts the. A plasmid is a small, extrachromosomal dna molecule within a cell that is physically separated from chromosomal dna and can replicate independently. Cloning a specific gene modern genetic analysis ncbi. The designation puc is derived from the classical p prefix and the abbreviation for the university of california, where early work on the plasmid series had been conducted. 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. In the first article in this series, we talked about how origins of replication ori control plasmid replication and copy number. The puc18 puc19 sequence is stored as a pdffile on the genaxxon webpage. Bacterial plasmids can not accept dna strands larger than 5000 base pairs, hence they are restricted to cloning dna. Pdf on jul 16, 2018, jens staal and others published engineering a minimal 1185 bp cloning vector from a puc18 plasmid backbone with an extended.
The molecule is a doublestranded circular dna 2686 base pairs in length. For detection of puc18 b, lane 3, a radiolabeled probe for the bla region was used. The purpose of this protocol is the isolation of plasmid dna from bacteria. Purification and identification of plasmid dna g rowth of e. View lab report transformation lab from bsc 1011l at florida international university. Vectors puc18 and puc19 are small highcopy number plasmids.
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. Aliquots of each transformation were selected on the appropriate antibioticagar plates and the low. Expression vector with lacz as an insertional detection marker. Lbagar plates with ampicillin for puc18 control transformations, see preparation of media on. Nov 26, 2014 today i would like to introduce you to puc18, a plasmid most noted for its high copy number. Experiment 2 plasmid dna ppasmid, restriction digestion. My lab students digested a recombinant puc18 plasmid with ecor1 restriction endonuclease.
The puc18 plasmid structure consists of the ampicillin resistance gene, a gene. How many puc18 plasmid dna molecules will be there in 10 ng. The puc plasmids are actually cole1 variants that lack the rop gene. Transformation summary for the puc18 control plasmid. Plasmid was created and is maintained by the dfhcc dna resource core at harvard medical school. Combining the two digested fragments in one tube allowed for the kanamycin. View homework help exercise 9 lab report from bsc 1010l at florida international university. Partitioning is strictly controlled for low copy, but loose for high copy 8. Lacz and pu cmini indic ate the location of the rn aii transcr ipt, which is i mporta nt for plasmi d replicat ion 5 but not in cluded in t he pmb1 ori anno tation.
Plasmid replication requires host cell functions 9. Highlights purified by chromatography using proprietary patented technology more than. Plasmid puc18minitn7tplppalpr was generated by am plification of the. The molecule is a small doublestranded circle, 2686 base pairs in length, and has a high copy number. Welcome to vector database vector database is a digital collection of vector backbones assembled from publications and commercially available sources. As many as 500 copies of this plasmid may be present in a single e. A plasmid is a circular dsdna molecule a few hundred or thousand base pairs in circumference. Vector database is a digital collection of vector backbones assembled from publications and commercially available sources. In each lane, the substrate refers to the monomeric form of the plasmid, and the excision product refers to the product of sitespecific recombination that contains the teta region. 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. For assistance in designing your custom vector using the surevector cloning. Use text editor or plasmid mapping software to view sequence. Jun 08, 2004 for detection of puc18 b, lane 3, a radiolabeled probe for the bla region was used. Due to a small size puc18 enables successful cloning of large dna fragments.1391 1506 552 1404 1145 131 314 441 191 1154 497 1441 783 807 706 1488 934 385 999 687 1406 494 1066 898 940 557 58 1262 1474 1275 1076 439 1254 872 40 640 1045 1438 973 560 852 382