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. Instaclone pcr cloning kit 30 rxns k1214 from thermo fisher. 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. It is ideal for phosphorylated or nonphosphorylated dna fragments. Pcr cloning is a rapid method for cloning genes, and is often used for projects that require higher throughput than traditional cloning methods can accommodate.
Strataclone solopack competent cells and puc18 control plasmid. Bluntend cloning of pcr fragments was carried out using the clonejet pcr cloning kit fermentas, st. This pcr cloning kit contains an optimized 2x cloning master mix with a proprietary ligation enhancer and a linearized vector that uses a novel mechanism for. Pcrbased cloning from plasmids entered by karin holmberg, kevin janes janes lab protocols updated by cheryl borgman 020817 3 excise the band on the lucite gel holder with a clean razor blade. These singlestranded aoverhangs are not compatible with the strataclone blunt pcr cloning vector arms. Can the cloning kit be used with inserts containing 5. Figure 1 in the qiagen pcr cloning kit handbook shows the pdrive cloning vector map. The stratagene pcrscript amp cloning kit is a polymerase chain reaction pcr cloning method that can be performed in 1 hour without adding bases to the primers. The topo xl2 complete pcr cloning kit is supplied with the purelink quick gel extraction and pcr purification combo kit for added flexibility allowing for faster pcr purification when inserts are smaller, while primers and dntps still need to be removed. Any other blunt or stickyend dna fragment can be cloned. Compare instaclone pcr cloning kit 30 rxns k1214 from thermo fisher scientific on.
The kit must produce 107 colonies from 250 ng of the exactstart rna control template when using epicentres transformax ec100 electrocompetent e. Use the pgemt vectors for general pcr cloning and the ptarget vector when you need to express the insert in mammalian cells. Msds documents are not included with product shipments. This kit includes the pfuultra ii fusion hotstart dna polymerase. Clones were selected on lb agar plates supplemented with 250 gml carbenicillin duchefa. Automatically annotate plasmid maps and expression vectors. The cells are provided in a convenient singletube transformation format. 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. Rapid and efficient cloning of large pcr products, up to. While this technique is generally routine, its applicability may not be sufficient to generate a desired target molecule from two separate regions involving intron exon boundaries. The topo xl pcr cloning kit has been optimized for topo cloning of long 310 kb pcr products. Reactions were chilled on ice for 1 minute and transformed into 100 l of chemocompetent e. Sticky end pcr cloning zeng, 1998 that allows one to generate sticky end by using standard pcr method is described below.
Efficient ligation occurs with vector arms charged with topoisomerase i on one end and a lox p recognition sequence on the other end. Cloning and screening of large pcr products is faster and easier using the copycontrol pcr cloning kit than by other commonly used methods. In this method, two pairs of pcr primers are designed and are amplified in two different reactions. Protocol for cloning insert into as2 series plasmids sticky end pcr method introduction. Use crystal violet instead of ethidium bromide etbr to visualize the pcr for gel isolation to avoid dna nicks. Map for the strataclone pcr cloning vector pscaampkan. Description ta cloning kit designed for cloning directly from a pcr reaction. The qiagen pcr cloning kit and box i of the qiagen pcr cloningplus kit containing ligation master mix and pdrive cloning vector should be stored at 20c in a constanttemperature freezer or at 70c immediately upon receipt. The strataclone pcr cloning kit allows highefficiency, 5minute cloning of pcr products, using the efficient dna rejoining activity of dna topoisomerase i and the dna recombination activity of cre recombinase. If using the regular topo kits, here are some tips to improve efficiency. 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. The strataclone pcr cloning kit was used to clone pcr fragments from a common fluorescent protein 664 bp, gammaglobin 1. Instaclone pcr cloning kit 30 rxns from thermo fisher scientific.
Thermo scientific clonejet pcr cloning kit is an advanced positive selection system for highefficiency cloning of pcr products generated with any thermostable dna polymerase. Box ii of the qiagen pcr cloningplus kit containing qiagen ez competent cells should be stored at 70c or. Rapid and efficient cloning of large pcr products, up to 15. The positions of the t7 and sp6 promoter sites and the m forward and reverse sequencing primer binding sites are provided. For these situations, the generation of fulllength complementary dnas from two partial genomic clones. Thermo scientific clonejet pcr cloning kit is an advanced positive selection system for highefficiency cloning of pcr products generated with any. Geneious prime is connected to plasmapper which allows you to automatically highlight plasmids with common promoters, terminators, cloning sites, restriction sites, reporter genes, affinity tags, selectable marker genes, replication origins and open reading frames. Pcr amplification of target molecules involves sequence specific primers that flank the region to be amplified. What links here related changes upload file special pages permanent link.
Pcr cloning is a rapid method for cloning genes, and is often used for projects that require higher throughput than. Qiagen pcr cloning handbook 012015 5 storage the qiagen pcr cloning kit and box i of the qiagen pcr cloningplus kit containing ligation master mix and pdrive cloning vector should be stored at 15 to 30c in a constant temperature freezer or at 70c immediately upon receipt. 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. Pcr cloning vector mix, strataclone pcr cloning kits agilent.
The enzyme adds a single, unpaired residuepreferentially an adenosyl residue to each 3 end of a doublestranded amplified product. Strataclone pcr cloning kit from stratagene biocompare. Rapid and efficient cloning of large pcr products, up to 15 kb, using the new copycontrol pcr cloning kits haiying grunenwald, epicentre preparation cloning screening table 1. Cloning of pcr products is important for most, if not all, researchers working in the life sciences. The strataclone pcr cloning kit is designed for efficient cloning of pcr fragments. Ta cloning is a subcloning technique that avoids the use of restriction enzymes and is easier. Read below to learn about how to achieve seamless cloning results via topoisomerase cloning, slic, and gibson. Pcr cloning differs from traditional cloning in that the dna fragment of interest, and even the vector, can be amplified by the polymerase chain reaction pcr and ligated together, without the use of restriction enzymes. Oct 09, 2006 the strataclone pcr cloning kit is designed for efficient cloning of pcr fragments. Specific pcr products of polymerase chain reaction pcr and ligated together, without the use of restriction enzymes.
Map for the strataclone blunt pcr cloning vector pscbampkan 6. Protocol for exactstart fulllength cdna library cloning kit. This pcr cloning kit contains an optimized cloning mix containing a proprietary ligation enhancer and a linearized vector that uses a novel mechanism for. Topoisomerasebased cloning saves time and money with simple primer design, no pcr cleanup required, and an easy 3step process. May 20, 2014 nebnext enzymatic methylseq kit workflow duration. Topo ta cloning kits supplied with the purelink quick plasmid miniprep. 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. Pcr cloning is a method in which doublestranded dna fragments amplified by pcr are ligated directly into a vector.
Pcrscript amp cloning kit 3 introduction the stratagene pcrscript amp cloning kit is a polymerase chain reaction pcr cloning method that can be performed in 1 hour without adding bases to the primers. Assuming you are amplifying from plasmid dna rather than from genomic dna or a cdna library, roughly 1821bp is usually sufficient to give specificity and to also be compatible with a standard pcr reaction. This pcr cloning kit contains an optimized 2x cloning master mix with a proprietary ligation enhancer and a linearized vector that uses a novel mechanism for background colony suppression to give a low background. Tvector cloning, or ta cloning, is a convenient method for cloning pcr products generated with taq dna polymerase. Purchase your selection instantly, save it or email it to a purchasing agent. These vectors are ready to use in ligation reactions. Pcr cloning offers some advantages over traditional cloning which relies on digesting doublestranded dna inserts with restriction enzymes to create compatible ends, purifying and isolating sufficient amounts, and ligating into a similarly treated vector of choice see insert.
I have extracted mrna from cells, converted it into cdna using quantitect reverse transcription kit. This kit utilizes the 3adenosine overhangs produced during amplification with taq. Manufacturers commonly sell ta cloning kits with a wide range of prepared vectors that. All components of the exactstart fulllength cdna library cloning kit are free of. Because we are cloning an orf, we want to clone from the start codon atg to the stop codon tga, in this example. 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. Can i use the neb pcr cloning kit featuring pminit 2. Limited label license for strataclone pcr cloning kits. Available for both bluntend and ua cloning, the kits can clone both long and short amplicons. This technology is based on topoisomerase i from the. The qiagen pcr cloningplus kit provides readytouse ligation reactions, which contain linearized cloning vectors that carry u overhangs at each 3 end.
The high quality ta cloning vector ptz57rt is ready to use for efficient ligation with pcr products providing high cloning yields and low background. Overview of strataclone pcr cloning technology using the method summarized in figure 1, strataclone pcr cloning. The thermo scientific instaclone pcr cloning kit is a ta system for direct onestep cloning of pcr products with 3da overhangs 1. During the cloning process, the ends of the dna of interest and the vector have to.
Here the researcher will find readily reproducible methods for all the major aspects of pcr use, including pcr optimization, computer programs for pcr primer design and analysis, and novel variations for cloning genes of special characteristics or origin, with emphasis on longdistance pcr and gcrich template amplification. What sequencing primers can i use with the pdrive cloning. 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. Search for user documents, sdss, vector maps and sequences, application notes. Protocols for cloning without restriction enzymes or ligases. Assuming you are amplifying from plasmid dna rather than from genomic dna or a cdna library, roughly 1821bp is usually sufficient to give specificity and to also be compatible with a. The kit contains strataclone vector mix, strataclone cloning buffer, solopack pcr cloning competent cells, strataclone control insert and puc18 control plasmid.
There are many cloning methods that do not require restriction enzymes or ligases. Strataclone pcr cloning kit 3 introduction the strataclone pcr cloning kit allows highefficiency, 5minute cloning of pcr products, using the efficient dna rejoining activity of dna topoisomerase i and the dna recombination activity of cre recombinase. The exactstart fulllength cdna library cloning kit is functiontested in a control reaction. Specific pcr products of strataclone blunt pcr cloning kit preprotocol considerations pcr enzyme selection the strataclone blunt pcr cloning system is designed for the cloning of blunt pcr products amplified by proofreading dna polymerases. We are using pcr products from the above method, cleaned up through an isopropanol precipitation step, as inserts for cloning into the pstblue1 vector. Instaclone pcr cloning kit 30 rxns k1214 from thermo. Topoisomerase technology topoisomerase technology requires no special primers and no ligases it is as easy as cloning comes. The strataclone combo pcr cloning kit contains 10 reactions of each. Once you have designed the cloning scheme, gathering all of the required reagents to get you from construct to expression and analysis is not trivial. Pcrbased cloning from plasmids entered by karin holmberg. This kit utilizes the 3adenosine overhangs produced during amplification.
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. Is it possible to clone the pcr product directly into an. Choochoo cloning tm kits are highly efficient directional pcr cloning kits for rapid, ligase and restriction enzymeindependent cloning of pcr products. When the template is a plasmid that encodes the ampicillin resistance gene, plate the transformation on kanamycin plates. The pdrive cloning vector contains the standard sequencing annealing sites for m forward and reverse, as well as t7 and sp6 primers. Cloneweaver helps you build a customized cloning kit with all of the items your cloning scheme requires. Common cloning applications and strategies thermo fisher.
Pcr enzyme recommendations for specific applications are listed in table 1. Nov 08, 2018 there are many cloning methods that do not require restriction enzymes or ligases. I am a beginner of cdna cloning for overexpression of my protein in cell line. Gelanalyze the pcr product for specificity and yield before cloning. The clonejet pcr cloning kit is compatible with all pcr buffers supplied by thermo scientific. This vector is available as part of a kit from novagen catalogue number 701914 which contains everything needed for the ligations and transformations, including the novablue. In addition, our strataclone ultra pcr cloning system is also available. Can the cloning kit be used for inserts that are not necessarily pcr amplicons. Jun 02, 2011 bluntend cloning of pcr fragments was carried out using the clonejet pcr cloning kit fermentas, st. A 500 bp pcr product incubated with the linearized vector in a 3. Fiveminute cloning of taq polymeraseamplified pcr products. Strataclone technology our strataclone pcr cloning systems exploit the combined efforts of two enzymes, dna. Our strataclone blunt pcr cloning kits and strataclone pcr cloning kits for ua cloning are available in both 10 and 20 reaction sizes.
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