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 puc19 multiple cloning site mcs is retained, however hincii, hindiii and psti are not unique in pfab. Use our dna sequences and maps tool to view the sequence files used to produce plasmid vectors, viral and bacteriophage maps from nebs catalog. Use experimental dna that is free of phenol, ethanol, salts, protein, and detergents to obtain maximum transformation efficiency. The features of this puc19 plasmids are as follows. Plasmid selection in escherichia coli using an endogenous. Coli plasmids different types principle nguyen thu giangipmb, vub. The fabi cassette was also inserted into the mcs of puc19 to obtain pucfa.
Today i would like to introduce you to puc18, a plasmid most noted for its high copy number. Plasmids pbr322 and puc8 pbr322 plasmid one of the first plasmids to be used in recombinant genetics was called pbr322. Please acknowledge the principal investigator, cite the article. This material is available to academics and nonprofits only. This resource is designed to educate all levels of scientists and plasmid lovers and serves as an introduction to plasmids, allowing you to spend. To see this sequence with restriction sites, features, and translations, please download snapgene or. All structured data from the file and property namespaces is available under the creative commons cc0 license. High copy number of the puc plasmid results from a romropsuppressible point mutation in rna ii. The creation of a sizemodified puc19 jennifer roger department of microbiology and immunology, ubc in order to study the exclusion effect observed when escherichia coli dh5. Bacillus megaterium high performance expression vectors pdf. The dna sequence of our mmp18 is 100% identical to the sequence of mmp18 from new england biolabs.
Download annotated snapgene files for a variety of commonly used genes and plasmid vectors. The multiple cloning site mcs is within the bgalactosidase gene. Unique restriction sites, so that the restriction enzymes can be used to cut the plasmid and dna of interest can be inserted into the plasmid. The molecule is a small doublestranded circle, 2686 base pairs in length, and has a high copy number. Discrepancies between sequencing results obtained by addgene and the original sequence provided by the depositor may be present. Transformation of bacteria with different plasmids objectives to understand the concept of dna as genetic material through the process of transformation. Pdf high copy number of the puc plasmid result from a. Fiveminute cloning of taq polymeraseamplified pcr products. Why are two different restriction enzymes used to cut the puc19 plasmid and the lux gene dna. Bacterial plasmids plasmids are defined as double stranded, extrachromosomal genetic elements that replicate independently of the host cell chromosome and are stably inherited. The following file naming structure is used to name the majority of these document files. It can be downloaded from the detaisl view of the puc18 puc19 product description. The focus of this study was the construction of a novel enlarged puc19 vector, pbart, which carries the rop and tetracycline resistance genes derived from pbr322. 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.
Use with snapgene software or the free viewer to visualize additional data and align other sequences. Bacillus megaterium classic expression vectors pdf. Lab 10 assignment lab 10 assignment molecular genetics. The puc19 plasmid 2,686 bp confers ampicillin resistance and complement defects in.
Plasmid sequence and snapgene enhanced annotations. Restriction enzyme mapping of puc19 given the map of the plasmid in figure 103, you should be able to predict the length of dna fragments that will result when these digests are completed. 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. Files are available under licenses specified on their description page. The cut sites for some restriction enzyme are indicated on the plasmid. A desktop resource, is to curate a onestop reference guide for plasmids. To test the conditions that make cells competent for use in dnamediated transformation. Transformation of competent cells with a recombinant plasmid carl estrella, merced college, merced, ca introduction. Plasmid and lambda vectors the fungal genetics stock center maintains a growing number of plasmids and lambda strains for the construction of gene libraries and the manipulation of cloned genes. A plasmid is a circular dsdna molecule a few hundred or thousand base pairs in circumference. Engineered plasmids can be used to clone genetic material of up to 10,000 base pairs, the amount of genetic material is limited by the size of the plasmid. Structural biochemistrydna recombinant techniquesplasmid. The bayou biolabs mmp18 sequence has a few differences from the mmp18 sequence reported in.
Why are two different restriction enzymes used to cut the. Isolation and characterization of plasmid dna part 3. 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. In the first article in this series, we talked about how origins of replication ori control plasmid replication and copy number. Cloning vectors cloning vectors are dna molecules that are used to transport cloned sequences between biological hosts and the test tube. Mattenley department of microbiology and immunology, ubc based on the observation that the transformation of escherchia coli was more efficient with puc19 than with the larger. The product summary sheet, or data card, includes details for how to use the product, as well as details of its formulation and quality controls. Thermo scientific puc19 vector is a small, high copy number, e. Construction of an enlarged puc19 vector with a rop gene. Lab 10 assignment molecular genetics and biotechnology. Plasmids capable of integration into the chromosome were earlier called episomes. For the bayou biolabs mmp18 sequence, please click here.
A vector is used to amplify a single molecule of dna into many copes. The journal is a bimonthly that publishes full articles, short. Sequence provided by depositing laboratory may be theoreticalpredicted or based on sangerngs sequencing results. Plasmid and lambda vectors fungal genetics stock center. The puc18 puc19 sequence is stored as a pdf file on the genaxxon webpage. What would have happened if only the hind iii enzyme was 2850178. How stable would puc19 be if i insert a big fragment 67kb on its mcs. It contains identical multiple cloning site mcs as puc18 vector except that it is arranged in opposite orientation. Use text editor or plasmid mapping software to view sequence. It also welcomes applications of plasmid biology to biotechnology and medicine, and of bioinformatics for studies of genomes. This page was last edited on 2 december 2019, at 12. It carries a 54 bp multiple cloning site polylinker.
This vector is not available from the plasmid repository and is provided here only as a reference. Helicobacter pylori strain atcc700392 encodes a methyl. Download as pptx, pdf, txt or read online from scribd. Download as ppt, pdf, txt or read online from scribd. The effect of increasing plasmid size on transformation efficiency in escherichia coli vicky chan, lisa f. The molecule is a doublestranded circle 4,361 base pairs in length 2. The effect of increasing plasmid size on transformation. The fabi cassette in pfab can be transferred to other pucderived plasmids using the aatii and alwni restriction sites. It has been constructed using the ampicillin resistance gene and the pmb1 origin of. These materials are made available in the same manner as fungal strains. 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 molecule is doublestranded circle, 2686 base pairs in length, and has a high copy number. Plasmid puc19 multiple cloning sites mcs for insertion of exogenous dna there is a stretch of dna within the betagalactosidase gene called polylinker or multiple cloning sites mcs.
The presence of mcs makes it possible to insert exogenous dna in the beta. Highlights purified by chromatography using proprietary patented technology more than. Plasmid welcomes topics such as horizontal gene transfer, including antibiotic resistance transfer, and molecular aspects of microbial ecology. Im planing to insert a big fragment into li for expresing some genes. Cloning is a method of recombining genes in order to take advantage of a bacterias native ability to recreate plasmids.
864 418 771 88 746 950 697 146 317 944 719 1578 1543 545 404 1264 525 1449 1089 1528 697 1091 1403 518 884 1294 1255 902 632 660 810 293 394 1228 690 586 883 779 319 943