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. 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. 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. These vectors are ready to use in ligation reactions. Topoisomerase technology topoisomerase technology requires no special primers and no ligases it is as easy as cloning comes. Jun 02, 2011 bluntend cloning of pcr fragments was carried out using the clonejet pcr cloning kit fermentas, st. 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. Protocols for cloning without restriction enzymes or ligases. 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. We are using pcr products from the above method, cleaned up through an isopropanol precipitation step, as inserts for cloning into the pstblue1 vector. Strataclone technology our strataclone pcr cloning systems exploit the combined efforts of two enzymes, dna. For these situations, the generation of fulllength complementary dnas from two partial genomic clones. This kit includes the pfuultra ii fusion hotstart dna polymerase.
Common cloning applications and strategies thermo fisher. When the template is a plasmid that encodes the ampicillin resistance gene, plate the transformation on kanamycin plates. Both pcr products are mixed in an equimolar ratio and purified using roche pcr cleaning kit or equivalent products. Topo ta cloning kits supplied with the purelink quick plasmid miniprep. 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. Limited label license for strataclone pcr cloning kits. Is it possible to clone the pcr product directly into an. The pdrive cloning vector contains the standard sequencing annealing sites for m forward and reverse, as well as t7 and sp6 primers. 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. 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. Cloneweaver helps you build a customized cloning kit with all of the items your cloning scheme requires. 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. Protocol for cloning insert into as2 series plasmids sticky end pcr method introduction.
There are many cloning methods that do not require restriction enzymes or ligases. 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. Taq dna polymerase catalyzes the nontemplate directed addition of an adenine residue to the 3. Ta cloning is a subcloning technique that avoids the use of restriction enzymes and is easier. The kit contains strataclone vector mix, strataclone cloning buffer, solopack pcr cloning competent cells, strataclone control insert and puc18 control plasmid. 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. These kits are available for both bluntend and ua cloning. Because we are cloning an orf, we want to clone from the start codon atg to the stop codon tga, in this example. Manufacturers commonly sell ta cloning kits with a wide range of prepared vectors that. This pcr cloning kit contains an optimized cloning mix containing a proprietary ligation enhancer and a linearized vector that uses a novel mechanism for.
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. Specific pcr products of polymerase chain reaction pcr and ligated together, without the use of restriction enzymes. Is it possible to clone the pcr product directly into an expression vector. This kit utilizes the 3adenosine overhangs produced during amplification. Can i use the neb pcr cloning kit featuring pminit 2. Instaclone pcr cloning kit 30 rxns k1214 from thermo. 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. Rapid and efficient cloning of large pcr products, up to 15. The thermo scientific instaclone pcr cloning kit is a ta system for direct onestep cloning of pcr products with 3da overhangs 1. They allow to clone any pcr fragment into any linearized vector at any location. Pcrbased cloning from plasmids entered by karin holmberg. Use crystal violet instead of ethidium bromide etbr to visualize the pcr for gel isolation to avoid dna nicks. 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. 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 high quality ta cloning vector ptz57rt is ready to use for efficient ligation with pcr products providing high cloning yields and low background. Search for user documents, sdss, vector maps and sequences, application notes. Can the cloning kit be used for inserts that are not necessarily pcr amplicons. Thermo scientific clonejet pcr cloning kit is an advanced positive selection system for highefficiency cloning of pcr products generated with any. The enzyme adds a single, unpaired residuepreferentially an adenosyl residue to each 3 end of a doublestranded amplified product. Pcr cloning is a method in which doublestranded dna fragments amplified by pcr are ligated directly into a vector. 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. Purchase your selection instantly, save it or email it to a purchasing agent. Sticky end pcr cloning zeng, 1998 that allows one to generate sticky end by using standard pcr method is described below. Instaclone pcr cloning kit 30 rxns from thermo fisher scientific. Instaclone pcr cloning kit 30 rxns k1214 from thermo fisher. Limited label license for strataclone ultra blunt pcr cloning kits. The clonejet pcr cloning kit is compatible with all pcr buffers supplied by thermo scientific.
I have extracted mrna from cells, converted it into cdna using quantitect reverse transcription kit. Available for both bluntend and ua cloning, the kits can clone both long and short amplicons. Traditionally, cloning was achieved by generation of a pcr fragment using primers designated to add restriction site overhangs at the 5 and 3 ends. Our strataclone blunt pcr cloning kits and strataclone pcr cloning kits for ua cloning are available in both 10 and 20 reaction sizes. Thermo scientific clonejet pcr cloning kit is an advanced positive selection system for highefficiency cloning of pcr products generated with any thermostable dna polymerase. 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. 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. In this method, two pairs of pcr primers are designed and are amplified in two different reactions. Choochoo cloning tm kits are highly efficient directional pcr cloning kits for rapid, ligase and restriction enzymeindependent cloning of pcr products. 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. Fiveminute cloning of taq polymeraseamplified pcr products. Pcr enzyme recommendations for specific applications are listed in table 1.
Map for the strataclone blunt pcr cloning vector pscbampkan 6. If using the regular topo kits, here are some tips to improve efficiency. Agilent strataclone cloning systems data sheet introduction our agilent strataclone kits make cloning your pcr products easier, faster, and more reliable than conventional pcr cloning methods. 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.
Pcr cloning is a rapid method for cloning genes, and is often used for projects that require higher throughput than. The qiagen pcr cloningplus kit provides readytouse ligation reactions, which contain linearized cloning vectors that carry u overhangs at each 3 end. 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. Automatically annotate plasmid maps and expression vectors. 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. Pcr amplification of target molecules involves sequence specific primers that flank the region to be amplified. What sequencing primers can i use with the pdrive cloning. 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. The positions of the t7 and sp6 promoter sites and the m forward and reverse sequencing primer binding sites are provided. 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. Gelanalyze the pcr product for specificity and yield before cloning.
Efficient ligation occurs with vector arms charged with topoisomerase i on one end and a lox p recognition sequence on the other end. Protocol for exactstart fulllength cdna library cloning kit. A 500 bp pcr product incubated with the linearized vector in a 3. All components of the exactstart fulllength cdna library cloning kit are free of. Strataclone solopack competent cells and puc18 control plasmid. 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. This kit utilizes the 3adenosine overhangs produced during amplification with taq. May 20, 2014 nebnext enzymatic methylseq kit workflow duration. Compare instaclone pcr cloning kit 30 rxns k1214 from thermo fisher scientific on. The exactstart fulllength cdna library cloning kit is functiontested in a control reaction. This technology is based on topoisomerase i from the. These singlestranded aoverhangs are not compatible with the strataclone blunt pcr cloning vector arms. Rapid and efficient cloning of large pcr products, up to. I am a beginner of cdna cloning for overexpression of my protein in cell line.
The kit must produce 107 colonies from 250 ng of the exactstart rna control template when using epicentres transformax ec100 electrocompetent e. Can the cloning kit be used with inserts containing 5. The strataclone combo pcr cloning kit contains 10 reactions of each. Strataclone pcr cloning kit from stratagene biocompare. Read below to learn about how to achieve seamless cloning results via topoisomerase cloning, slic, and gibson. 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.
In addition, our strataclone ultra pcr cloning system is also available. During the cloning process, the ends of the dna of interest and the vector have to. Nov 08, 2018 there are many cloning methods that do not require restriction enzymes or ligases. Oct 09, 2006 the strataclone pcr cloning kit is designed for efficient cloning of pcr fragments. Description ta cloning kit designed for cloning directly from a pcr reaction. Map for the strataclone pcr cloning vector pscaampkan. It is ideal for phosphorylated or nonphosphorylated dna fragments. Msds documents are not included with product shipments. 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. The topo xl pcr cloning kit has been optimized for topo cloning of long 310 kb pcr products. Overview of strataclone pcr cloning technology using the method summarized in figure 1, strataclone pcr cloning.
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. The strataclone pcr cloning kit was used to clone pcr fragments from a common fluorescent protein 664 bp, gammaglobin 1. 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. What links here related changes upload file special pages permanent link. The cells are provided in a convenient singletube transformation format. Any other blunt or stickyend dna fragment can be cloned. Box ii of the qiagen pcr cloningplus kit containing qiagen ez competent cells should be stored at 70c or. The strataclone pcr cloning kit is designed for efficient cloning of pcr fragments. Pcr cloning vector mix, strataclone pcr cloning kits agilent. Bluntend cloning of pcr fragments was carried out using the clonejet pcr cloning kit fermentas, st. 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. Clones were selected on lb agar plates supplemented with 250 gml carbenicillin duchefa. Use the pgemt vectors for general pcr cloning and the ptarget vector when you need to express the insert in mammalian cells. Topoisomerasebased cloning saves time and money with simple primer design, no pcr cleanup required, and an easy 3step process.
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