Student manual promega corporation molecular cloning is a basic technique used in a molecular biology labs. Molecular cloning, characterization and diagnostic. Taqdna polymerase forcolonypcr wasfrom genespin milano,italy. Strataclone pcr cloning kit 5 strataclone solopack competent cells the provided strataclone solopack competent cells express cre recombinase, in order to circularize the linear dna molecules produced by topoisomerase imediated ligation. Using this kit, i completed synthesis of a bluntend 0.
The pcr product was initially cloned into the psca vector strataclone pcr cloning kit. Cloning vectors and kits the cloning of a specific fragment of dna into a vector such as a plasmid is one of the foundational steps that enables additional characterization of that sequence. The strataclone pcr cloning kit agilent genomics was used to clone pcr products as required. Nucleospin plasmidand nucleospin gel and pcr cleanup kits were from machereynagel. Strataclone blunt pcr cloning kit from agilent technologies. The cells are provided in a convenient singletube transformation format. Pdf over the last decades, molecular cloning has transformed. Strataclone pcr cloning kit avoid including the sequences ctcctt or aaggga in the pcr primers. In our lab, we will use the strataclone pcr cloning kit from agilent technologies.
The method involves first carrying out the pcr with taq dna polymerase, which was done last week. Purchase of this product conveys to the purchaser only the nontransferable right under these patents to use the product for research use only by the purchaser. Strataclone ultra blunt pcr cloning kit 5 strataclone solopack competent cells the provided strataclone solopack competent cells express cre recombinase, in order to circularize the linear dna molecules produced by topoisomerase imediated ligation. Pdf molecular cloning using polymerase chain reaction, an. The stratagene pcmvscript pcr cloning kit is a polymerase chain reaction pcr cloning method that can be performed in 1 hour without adding bases to the primers. To summarize, the scientist performing the procedure digests both the gene of interest and the plasmid dna. When the template is a plasmid that encodes the ampicillin resistance gene, plate the transformation on kanamycin plates. Cloning, characterization and diagnostic performance of. Avoid including the sequences ctcctt or aaggga in the pcr primers. Mix the following components in a sterile microfuge tube. Strataclone pcr cloning kit 7 using plasmid dna as pcr template genomic dna, plasmid dna, or cdna may be used as template for pcr amplification prior to cloning. Strataclone pcr cloning kit agilent t echnologies, catalog number.
Due to the emergence of novel and efficient pcr reagents, cloning kits. Identification of the biosynthetic gene cluster of a. The thermo scientific clonejet pcr cloning kit is an advanced positive selection system for highefficiency cloning of pcr products generated with any thermostable dna polymerase. The clonejet pcr cloning kit is compatible with all pcr buffers supplied by thermo scientific. Unstained protein molecular weight marker for sdspage was from thermo scienti. The agilent strataclone pcr cloning kit enables highefficiency, 5minute cloning of pcr products at room temperature.
Pcr products were gel extracted qiaquick gel extraction kit, qiagen and cloned into a plasmid strataclone pcr cloning kit, stratagene, santa clara, ca, usa. Pdf 3 rapid amplification of cdna ends 3 race using. Design of primers for pcr reaction for quadplex grna cloning the scalability of the multiplex grna cloning kit allows for. Taq dna polymerase catalyzes the nontemplate directed addition of an adenine residue to the 3. The easya onetube rtpcr system can readily detect rna targets of 0. Association studies to examine the association of exon skipping and spinosad resistance, 50 male pupae of the spinsel strain were. May 29, 2009 for rt pcr cloning of ndpglucose dehydratase ngdh in s.
Zero blunt pcr cloning kit thermo fisher scientific. Efficient cloning of pcr products into a plasmid for sequencing and. For qiagen pcr cloning kit qiagen pcr cloningplus kit pdf 771kb english format file size language download get adobe reader contact qiagen. Additionally, any other dna fragment, either blunt or stickyend, can be. For rtpcr cloning of ndpglucose dehydratase ngdh in s. This pcr cloning kit contains an optimized cloning mix containing a proprietary ligation enhancer and a linearized vector that uses a novel mechanism for background colony suppression to give a low background. This kit utilizes the 3adenosine overhangs produced during amplification. They use a pcr topo vector with covalently bound topoisomerase i for fast cloning and recombinants. The pcr fragments can be generated by taq dna polymerase or other high fidelity dna polymerases.
Cloning efficiency is optimized, however, by implementing the following primer design considerations. These singlestranded aoverhangs are not compatible with the strataclone blunt pcr. Identification of the biosynthetic gene cluster of a500359s. If the pcr product is amplified from a plasmid template, then it needs to be gelpurified to reduce background. Specific pcr products of pcr cloning kit contains a novel, readytouse positive selection cloning vector pjet1. Topo ta cloning kits are designed for cloning pcr products directly from a pcr reaction in just 5 minutes 1. Biochemical and spectroscopic properties of brucella. Pcr cloning with lowno background a 500 bp pcr product incubated with the linearized. May 30, 2007 using this kit, i completed synthesis of a bluntend 0. The addition of a a by taq dna polymerase is not required and has no effect on cloning efficiency. Pdf molecular cloning using polymerase chain reaction. The vector contains a lethal restriction enzyme gene that is disrupted by ligation of a dna insert into the cloning site. Molecular cloning using polymerase chain reaction, an educational. At least three different clones of the insert were sequenced to rule out sequencing errors.
This pcr product was cloned into the psca vector using the strataclone pcr cloning kit stratagene, following the manufacturers instructions, and sequenced on both strands. Strataclone ultra blunt pcr cloning kit 7 preprotocol considerations pcr primer design no specific primers are required for the strataclone ultra blunt pcr cloning system. Thermo scientific clonejet pcr cloning kit, also available with dh10b cells k123120, is an advanced positive selection system for highefficiency cloning of. Bluntended pcr products generated with a proofreading enzyme e. Thermo scientific clonejet pcr cloning kit for 20 reactions. A total of 10 colonies per orf were analyzed, and the plasmid inserts were. Equally fast, a coworker cloned an 82 bp dna pcr product, indicating that the size of insert is not likely to affect your success and time schedule.
By simply incubating on ice, the ends of a pcrgenerated dna fragment can be precisely fused into a dna vector with 6 bp or more overlapping. Topo ta cloning kit, dual promoter, with pcriitopo. The strataclone pcr cloning kit is designed for efficient cloning of pcr fragments. Specific pcr products of en qiagen pcr cloning handbook. The kit also allows direct cloning from amplification reactions without purification, and works well whether or not the primers used in the pcr possess 5. Tvector cloning, or ta cloning, is a convenient method for cloning pcr products generated with taq dna polymerase. Any other blunt or stickyend dna fragment can be cloned.
The reagents for both 1ststrand cdna synthesis and pcr amplification are combined in one tube. Dna polymerase, thermo scientific dreamtaq dna polymerase or other thermostable dna polymerases. Pcr cloning vector mix, strataclone pcr cloning kits agilent. An introduction to pcr cloning pcr products generated using a nonproofreading dna polymerase which lacks 3 to 5 exonuclease activity, such as taq dna polymerase, are left with a single templateindependent nucleotide, deoxyadenosine a, at the 3.
Association studies to examine the association of exon skipping and spinosad resistance, 50. They allow to clone any pcr fragment into any linearized vector at any location. There are several pcr cloning kits available some of which are shown in table 2. Pcr products were electrophoresed in an agarose gel and the cdna band was purified from the gel using the strataprep dna gel extraction kit stratagene.
The strataclone pcr cloning kit allows highefficiency, 5minute cloning of pcr products at room temperature, using the efficient dna rejoining activity of dna topoisomerase i and the dna recombination activity of cre recombinase. Establishment of a quantitative realtime pcr for the. A high efficiency system for cloning bluntended pcr. Characterization of plasmid ppo1 from the hyperacidophile.
Strataclone vector mix, strataclone cloning buffer, strataclone control insert. Pcrbased cloning from plasmids entered by karin holmberg. Description the genscript cloneez pcr cloning kit is designed for the quick pcr cloning, it is especially powerful in highthroughput cloning of pcr products into any destination vector effectively by sidestepping the tedious and limiting tasks such as selecting proper restriction enzymes, phosphatases, or ligases. Pcr primer design when using topoisomerase cloning kits e. The zero blunt pcr cloning kits are shipped on dry ice. Strataclone blunt pcr cloning vector pscbampkan, uses the dna. Add 20 l of the bacteria to the ligation and mix gently by pipetting. The cdna obtained was subjected to nested pcr with supertaq plus dna polymerase ambion using the primers listed in table 1. Vectors such as the pgem series contain multiple cloning sites to excerpt fragments generated by digestion with specific restriction enzymes. We used undiluted pcr reaction mix 2 ul to clone pcr fragments. For qiagen pcr cloning kit qiagen pcr cloningplus kit pdf 771kb english. Apobec3mediated hypermutation of retroviral vectors. Strataclone pcr cloning kit from stratagene biocompare. Increase the speed and efficiency of your pcr product cloning today with the agilent strataclone pcr cloning kit.
The kit contains strataclone vector mix, strataclone cloning buffer, solopack pcr cloning competent cells, strataclone control insert and puc18 control plasmid. The clonejet pcr cloning kit is an advanced positive selection system for the highest efficiency cloning of pcr products generated with. Download a pdf containing pricing for our full product list. Gelanalyze the pcr product for specificity and yield before cloning. Cloning, characterization and diagnostic performance of the. Taqamplified pcr products contain a single adenosine a nucleotide overhang on the 3 ends. Jun 21, 2019 to insert the cdna for dianthin into the expression vector. A xhoihindiii fragment was released from the pcr vector and ligated to a hindiiixbai fragment from pegfp1 clontech, mountain view, ca. Strataclone pcr cloning kit from agilent technologies. Qiagen and cloned using a strataclone pcr cloning kit stratagene. Genscripts proprietary cloneez seamless cloning technology is a cuttingedge, recombinationbased cloning technology that can accurately and efficiently assemble genes and gene fragments in any combination of sequences. The thermo scientific instaclone pcr cloning kit is a ta system for direct onestep cloning of pcr products with 3da overhangs 1.
Strataclone pcr cloning kit 5 solopack pcr cloning competent cells the provided solopack pcr cloning competent cells express cre recombinase, in order to circularize the linear dna molecules produced by topoisomerase imediated ligation. Pcr enzyme recommendations for specific applications are listed in table 1. Pcr cloning with lowno background a 500 bp pcr product incubated with the linearized vector in a 3. Note do not use taq dna polymerase, or enzyme blends containing predominantly taq dna polymerase, to amplify fragments for cloning using the strataclone blunt pcr cloning kit. Choochoo cloning tm kits are highly efficient directional pcr cloning kits for rapid, ligase and restriction enzymeindependent cloning of pcr products. It allows simple and quick cloning of any pcr amplicon. Biochemical and spectroscopic properties of brucella microti. Molecular cloning using polymerase chain reaction, an educational guide for cellular engineering.
In this manual, we include a protocol for isolating the luciferase gene from. The presence of one of these sequences in the primer creates a topoisomerase ibinding site ccctt, or tcctt in the pcr product. This amplicon was cloned using the strataclone pcr cloning kit. Clonejet pcr cloning kit is an advanced positive selection system for highefficiency cloning of pcr products generated with any thermostable dna polymerase. Qiagen pcr cloningplus kit transformation protocol. Easya highfidelity pcr cloning enzyme picomaxx highfidelity pcr system routine enzymes paq5000 dna polymerase other pcr enzymes pcr enhancers reverse transcription accuscript highfidelity rt accuscript pfuultra ii rtpcr kit affinityscript multiple temperature rt affinityscript onestep rtpcr kit sample preparation strataclone pcr. The template for the polymerase chain reaction pcr reaction was 1 ng of the plasmid. These kits are available for both bluntend and ua cloning. Reaction volume may be scaled up or down as necessary. Pcrbased cloning from plasmids entered by karin holmberg, kevin janes janes lab protocols updated by cheryl borgman 020817 2 reducing the volume allows the purified amplicon to be digested directly without concentrating it 5.
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