Where a primer is designed to overlap a restrictiondigested dna fragment, the 15bp overlap is counted from the cleavage site on the antisense dna strand as described in the clontech infusion user manual. For more detail refer to the gateway manual from the manufacturer. Phusion highfidelity dna polymerase high performance for. Most primers are 3555 bp, and we find quality control by mass spectroscopy to provide sufficient purity midland certified, midland, tx, usa. The commercial user friendly cloning kit new england biolabs, launched in 2003 makes use of a similar double enzymatic treatment. Intact genomics propriety igfusion cloning technology is a simple, rapid and highly efficient cloning kit which allows to directly clone any pcr products to any linearized expression vector at any site. However, if you are amplifying from a pool of cdna, the amount of template dna required depends on the relative abundance of the target message in your mrna population. Advancing user cloning into simpleuser and nicking cloning. Infusion cloning technology makes it easy to clone your smarter cdna library into any location within any vector. Sleight sc, bartley ba, lieviant ja, sauro hm 2010 infusion biobrick assembly and reengineering. Sep 11, 2017 infusion hd cloning kit user manual b. Recombinational cloning using gateway and infusion cloning.
Having previously and successfully used the in fusion cloning kit im having real issues with my current round of cloning. Infusion hd multipleinsert cloning protocolataglance 121416 takara bio usa, inc. Infusion hd ecodry cloning kits are designed for fast and convenient, directional cloning of one or more fragments of dna into any vector. Pcr amplification of target fragment for most dna polymerases, 100 pg1 ng of plasmid dna is enough to use as a pcr template. I have a number if 2kbish fragments that im trying to insert into the. The infusion cloning reaction, which takes as little as 15 minutes, is specific and directional, ensuring an exceptionally high rate of cloning accuracy in all applications. The infusion cloning products allow ligationindependent cloning of pcr products into any vector, at any site of linearization. Introduction infusion hd ecodry cloning kits are designed for fast and convenient, directional cloning of one or more fragments of dna into any vector. In this cloning technique, the pcr product is treated with user enzyme mix, a mixture of udg and dna glycosylaselyase endo viii the exact proportions are not disclosed by the company. In this chapter, we present a general protocol for converting any vector into a user compatible vector, together with protocols for both user cloning. Experience the thrill of the chillcloning in less than 20 minutes clone any insert, at any site within any vector restriction enzyme, phosphatase and ligase free system. Infusion advantage pcr cloning kit user manual infusion advantage pcr cloning kits are designed to join multiple pieces of dna that have 15 bases of homology at their linear ends. Nebuilder hifi dna assembly offers several advantages over infusion hd.
Infusion smarter directional cdna library construction kit user manual pt51471 notice to purchaser clontech products are to be used for research purposes only. Snapgene was the first software to simulate this procedure. The slic, gibson, cpec, and slice assembly methods and. Infusion cloning procedure for spincolumn purified pcr fragments x.
Details on multifragment infusion cloning can be found in our tech note infusion multiple fragment cloning. Between 25100 ng of vector was mixed at a ratio of 1 mole vector to 2 moles of each additional dna segment in 10 l of water and added to one well containing the lyophilized infusion reaction mix. Pcr primer design after digestion, purify the linearized vector using a dna gel extraction kit for high purification efficiency to increase the chances of a successful fusion reaction. The preference of ligation independent cloning types gibson, in fusion, licpcr cloning etc. Infusion advantage pcr cloning kit protocolataglance pt40652 please read the infusion advantage user manual pt40651 before using this protocolataglance. Infusiontm drydown pcr cloning kit protocolataglance. User manual store the master mixture and positive controls at 20. Troubleshooting guide infusion hd cloning kit user manual takara bio usa, inc. If your vector and pcr product differ greatly in size e. I used the recombination kit of clontech infusion, transform the bacteria but had no colonies.
Is anyone familiar with experience of infusion cloning. The cold fusion master mix contains a proprietary blend of enzymes that prepares dna fragments ends for. Infusion smarter directional cdna library construction kit. Infusion cloning pcr is done with only one round of pcr with one set of primers. Infusion hd ecodry cloning kit user manual takara bio. The cold fusion master mix contains a proprietary blend of enzymes that prepares dna fragments ends for sequencedirected alignment. Infusion hd ecodry cloning kit user manual 080318 takara bio usa, inc. The gibson assembly master mix and gibson assembly cloning kit are warranted to perform according to specifications stated on the certificate of analysis.
Please read the user manual pt37541 before using this protocolataglance. F, enda1, supe44, thi1, reca1, rela1, gyra96, phoa. After incubation, the infusion reaction was diluted with te. List of components all infusion hd cloning kits contain 5x infusion hd enzyme premix, linearized puc19 control vector 50 ng. Infusion hd cloning kit user manual protocol online.
Having previously and successfully used the infusion cloning kit im having real issues with my current round of cloning. While these systems do allow rapid cloning, they depend on specific sequences within the target vectors and, thus, restrict the user to a particular series of vectors. Highthroughput cloning of functional elements in the. In this chapter, we present a general protocol for converting any vector into a usercompatible vector, together with protocols for both user cloning. Pcr based, universal, suitable for multiple fragment cloning join the revolution in new cloning technologies. May 16, 2018 where a primer is designed to overlap a restrictiondigested dna fragment, the 15bp overlap is counted from the cleavage site on the antisense dna strand as described in the clontech in fusion user manual. Cloning and competent cells 4 infusion mutagenesis protocol overview please see the infusion hd cloning kit user manual for detailed instructions. Typically, infusion reactions work well with 100 ng of a 45 kb linearized vector plus 50 ng of a 1 kb pcr product. Aug 28, 2014 with in fusion hd cloning plus, there is no requirement for an additional, separate pcrbased mutation kit as both cloning and mutagenesis can be carried out with the same system. Bd infusion method efficiently clones a range of insert sizes.
Jan 12, 2006 while these systems do allow rapid cloning, they depend on specific sequences within the target vectors and, thus, restrict the user to a particular series of vectors. The cornerstone of infusion cloning technology is our proprietary infusion. Genbuilder dna assemblyseamless cloningrecombination. The phusion highfidelity dna polymerase should be pipetted carefully and gently as the high glycerol content 50% in the storage buffer may otherwise lead to pipetting errors. A typical use for this technology would be to clone pcr products into vectors, without the use of restriction enzymes, ligase or phosphatase. Infusion reactions were set up according to the clontech infusion kit user manual. If your pcr product contains multiple bands, gelpurify instead using the nucleospin gel and pcr cleanup kit.
It therefore seemed obvious to create a simplified version of user cloning in a way that makes it less laborious. Prepare plasmid dna using any commercially available miniprep kit or standard. Nfusion user manual infusion hd cloning kit user manual 1290 terra bella avenue, mountain view, ca 94043, usa. Student manual promega corporation molecular cloning is a basic technique used in a molecular biology labs. I used the recombination kit of clontech in fusion, transform the bacteria but had no colonies. Cold fusion cloning kit with competent cells system biosciences. In this manual, we include a protocol for isolating the luciferase gene from. Streamlining vector construction with cold fusion cloning.
All primers designed in direct accordance with the instructions. Jun 10, 2012 demonstration video showing how to simulate clontechs in fusion cloning using snapgene molecular biology software from gsl biotech. Matchmaker chemiluminescent coip system user manual protocol no. Using our proprietary in fusion enzyme, this kit rapidly generates precise constructs with inserts in the correct orientation. Because of this, and because of the exhaustive amounts of novel.
Biobrick assembly using the infusion pcr cloning kit. Infusion hd cloning kit user manual 102518 takara bio usa, inc. Pushing the bounds of innovation, the genbuilder technology allows for seamless fusion of one to twelve gene fragments with the highest efficiency. The clontech infusion pcr cloning system allows you to fuse of the ends of the pcr fragment to the homologous ends of a linearized vector.
Genscripts genbuilder dna assembly technology takes recombinationbased cloning to the next level. Due to the nature of the phusion highfidelity dna polymerase, the optimal. Cloning and competent cells 4 in fusion mutagenesis protocol overview please see the in fusion hd cloning kit user manual for detailed instructions. Having previously and successfully used the infusion cloning kit im having real issues with. Using our proprietary infusion enzyme, this kit rapidly generates precise constructs with inserts in the correct orientation.
The preference of ligation independent cloning types gibson, infusion, licpcr cloning etc. Page 1 of 7 please read the infusion hd cloning kit user manual before using this protocolataglance. By use of this product, you accept the terms and conditions outlined in the licensing and warranty statement contained in this user manual. Intact genomics propriety igfusion cloning technology is a simple, rapid and highly efficient cloning kit which allows to directly clone any pcr products to. System biosciences 2014 instruction manual of cold fusion cloning kit. This abbreviated protocol is provided for your convenience, but is not intended for firsttime users. Pcr cleanup kit according to manufacturers instructions. For the infusion reaction, a linearized vector is mixed with one or more pcr products that have overlapping ends. A takara bio company page 19 of 30 smarter race 53 kit user manual in general, the ld pcr method is more direct and less subject to complications or artifacts.
The 3 and 5 regions of homology are generated by adding 15 bp extensions to both pcr primers that precisely match the ends of the linearized vector. Infusiontm drydown pcr cloning kit user manual the infusiontm pcr cloning kits are designed for highthroughput cloning of pcr products without the need for restriction enzymes, ligase, or bluntend polishing. Biobrick assembly using the infusion pcr cloning kit article pdf available in methods in molecular biology clifton, n. Bd infusion cloning was performed using 100 ng linear vector and 50 ng of each of the pcr products indicated. With infusion hd cloning plus, there is no requirement for an additional, separate pcrbased mutation kit as both cloning and mutagenesis can be carried out with the same system. Demonstration video showing how to simulate clontechs infusion cloning using snapgene molecular biology software from gsl biotech. Nebuilder hifi dna assembly benefits over in fusion hd neb. Is anyone familiar with fusion hd cloningprimer design. User cloning is suitable not only for everyday and highthroughput cloning but also for the onestep construction of complex dna constructs, which can be achieved in a variant called user fusion. Introduction in fu s ion hd cloning kit s are designed for fast, directional cloning of one or more fragments of dna into any vector.
Snapgene simplifies infusion cloning by automating the primer design. Mar 27, 2007 the commercial user friendly cloning kit new england biolabs, launched in 2003 makes use of a similar double enzymatic treatment. Infusion hd cloning kit user manual 011614 clontech laboratories, inc. Matchmaker chemiluminescent coip system user manual. Clontechs infusion pcr cloning kit is a flexible system designed to be compatible with any desired vector. Cold fusion cloning is a quick and easy way to place an insertor multiple inserts simultaneouslyinto a linearized vector. Infusion hd multipleinsert cloning protocolataglance. Clontechs infusion cloning is a remarkably versatile method for creating seamless gene fusions. No other warranty is made, whether express or implied, including any warranty of merchant ability or fitness for a particular purpose.