Excerpts and links may be used, provided that full and clear credit is given to promega corporation with appropriate and specific direction to the original content. Find more similar flip pdfs like gblocks gene fragments cloning protocols. Ta cloning also known as rapid cloning or t cloning is a subcloning technique that avoids the use of restriction enzymes and is easier and quicker than traditional subcloning. The pgemt vectors are a popular choice for general pcr cloning. T overhangs at the insertion site greatly improve the efficiency. Check pages 1 14 of gblocks gene fragments cloning protocols in the flip pdf version. The pgemt easy vector systems are convenient systems to clone pcr products generated by certain thermostable polymerases. Apr 03, 2018 ligation independent cloning was reported, but with the use of an intermediary e. Promega offers a wide range of tools to facilitate cloning. The universal ta cloning method is thus both convenient and laborsaving. Preferential ligation during tacloning of multitemplate. Bsaisite removed from the ampicillinresistant gene allows for the cloning of golden gate assembly modules fast cloning experiments with 5minute ligation step. A overhangs taq polymerase amplified to topo vector with t overhangs competent cells.
Recent advances in universal ta cloning methods for use in. Moreover, tvectors have proved useful in systematic evolution of ligands by exponential enrichment selex yu et al. Ta cloning failure with pgemt easy vector and jm109. Ii analyze the plasmid dna by restriction analysis or ta. Further information on cloning in plasmid vectors can be found in promega s protocols and applications guide, third edition, chapter 3, and. Thermo scientific rapid dna ligation kit enables fast stickyend or bluntend dna ligation in only 5 minutes at room temperature. Cloning was carried out with both pgemt vector trometer, perkinelmer life and analytical sciences, boston, ma using system cat.
The pcr amplified dna fragment including the full length rar. The kit contains t4 dna ligase and a speciallyformulated 5x rapid ligation buffer optimized for fast and efficient dna ligation. Preferential ligation during tacloning of multitemplate pcr. Generating a custom tacloning expression plasmid for. The thermo scientific dreamtaq green buffer 10x supports direct gel loading of pcr products.
Aversatile zero background tvector system for gene cloning. If you have not amplified the product with re sites in mind, then it is a good idea to use either ta overhang vectors many in the market or simply use the pgem vectors supplied by promega. The pgemt vector systems are convenient systems to clone pcr products generated by certain thermostable polymerases. Ta cloning plasmids contain a single thymidine residue at. For this purpose, the protein coding sequence is cloned into a suitable expression vector and transferred into cells. Ligation conditions followed the manufacturers protocol optimized for high transformant yields, meaning 18 h ligation at 4 c for the pgemt vector, and 30 min. T and pgemt easy vector systems technical manual promega. Pcr cloning with bluewhite selection and easy insert excision. Produce pcr products using taq polymerase and your own protocol. Recently, a gateway cloning system was introduced as well. Further information on cloning in plasmid vectors can be found in promega s protocols and applications guide, third edition, chapter 3. Ta cloning topo ta cloning provides a highly efficient, 5minute, onestep cloning strategy topo cloning for the direct insertion of. Tvector cloning, or ta cloning, is a convenient method for cloning pcr products generated with taq dna polymerase.
Figure 1 summarizes the activities of the cloning enzymes. May 01, 2011 cloning was carried out with both pgemt vector system cat. A novel series of highefficiency vectors for ta cloning. Technical manual pgemt and pgemt easy vector systems instructions for use of products a60, a80, a3600 and a3610. In pcrt 2,728 bp for ta cloning, toverhang vector for cloning of datailed. The improved tvector system takes advantage of the restriction enzyme xcmi to generate a toverhang after digestion and. The pgem t and pgem t easy vector systems are convenient systems for the cloning of pcr products. Topoisomerase ibased, 5minute ligation of pcr products with 3.
Easy cloning of all pcr products, including blunt and ta ends. The technique relies on the ability of adenine a and thymine t complementary basepairs on different dna fragments to hybridize and, in the presence of ligase. The enzymes highlighted in this second enzyme resource guide, cloning enzymes, are those important in nucleic acid cloning procedures. A novel series of highefficiency vectors for ta cloning and bluntend. Choose from a range of dna, rna and protein molecular weight markers. Updated to allow for in vitro transcription with both sp6 and t7 promoters. Promega corporation 2800 woods hollow road madison, wi 537115399 usa. Gblocks gene fragments cloning protocols was published by on 20150516. Convenience and flexibility for cloning pcr products. The technique relies on the ability of adenine a and thymine t complementary basepairs on different dna fragments to hybridize and, in the presence of ligase, become ligated together. To be the most responsive supplier of biological reagents and reagent systems used in research and applied technology applications worldwide. Three options for removing the insert with a single digest bstzi, ecori or noti.
We evaluated the cloning efficiency of different size pcr products into three tvector cloning systems. Ta cloning kit dual promoter pcrii catalog number k205001, k205040, k206001, k206040, k2070 20, and k207040 pub. Tray cell hellma, mullheim, germany by absorbance at 260 nm with no. The two tracking dyes and a density reagent in the green buffer do not interfere with pcr performance and are compatible with downstream applications including dna sequencing, ligation, and.
K450001, invitrogen, ca usa in case of mixtures b3 and c3 table 1. Here we describe the development of a ta cloning system based on the nisincontrolled expression system nice plasmid pnz8148. However, several problems have been encountered using. Ta cloning relies on the complementary bases of adenine a and thymine t on different dna fragments to hybridize. The enzyme dna ligase can then be used to generate the. Ta cloning failure with pgemt easy vector and jm109 supercompetent cells from promega. Apr 23, 2019 design of plasmid vectors for pcr cloning. No ligase, postpcr procedures, or pcr primers containing specific sequences are required. Here, we report a zero background ta cloning system that provides simple and highef. When taq polymerase amplifies a piece of dna during pcr, the terminal transferase activity of taq adds an extra adenine at the 3. The pgemt and pgemt easy vectors are linearized vectors with a single.
Invitrogen topo ta cloning kits are designed for cloning pcr products amplified by taq dna polymerase, helpful tips which leaves an adenine at the 3. Ceuxci peuvent etre donc directement clones par simple ligation dans des. The pgemt easy vector systems are convenient systems to clone pcr products generated. The ta cloning vector was designed so that when linearized it has single 5. Bsaisite removed from the ampicillinresistant gene allows for the cloning of golden gate assembly modules. Heterologous expression and characterization of a high redox. The pgemt and pgemt easy vector systems are convenient systems for the cloning of pcr products. Topo pcr cloning solutions thermo fisher scientific. Generation of pcr products for ta cloning why use green. Fiveminute cloning of taq polymeraseamplified pcr products. These vectors are ready to use in ligation reactions. Promega corporation is a worldwide leader in applying biochemistry and molecular biology to the development of innovative, highvalue products for the life sciences.
Jul 08, 2019 unauthorized use andor duplication of this material without express and written permission from this promega corporation is strictly prohibited. Aversatile zero background tvector system for gene. I constructed three plasmid vectors for pcr cloning in this study fig. The pcr product can be ligated into this vector without the need for special restriction enzyme sites. Cloning protocols and applications guidea4 format promega. The pgemt and pgemt easy vector systems gave a high number of recombinants across a broad range of insert sizes 0. K450001, invitrogen, ca usa in case of mixtures b3 and c3. Cloning pcr products with pgemt and pgemt easy vectors. End the pcr reaction with a final 710 minute extension step. K451002 and k450002 refer to the manual supplied with the miniprep kit. The toverhangs at the insertion site greatly improve the efficiency of.
Ta cloning was the preferred strategy for cloning the poppcr products because their exact sequences were unknown. For cloning promoter sequences in order to assess their transcriptional regulatory functions. Molecular weight markers dna ladders rna ladders promega. Some thermostable dna polymerases, including taq, add a single nucleotide base extension to the 3. Invitrogen topo ta cloning kit for subcloning, without. Enzymes that modify nucleic acids provide the foundation for many molecular biology techniques. Ta cloning technology greatly simplifies traditional restriction and ligation cloning with a onestep cloning strategy that eliminates the need for any enzymatic modifications of the pcr product and not require the use of primers that contain restriction enzyme sites. Table 1 provides a list of the common applications of the six enzymes included in this guide. Cloning methods can be divided into two classes, depending on. Specific features of the in vivo functions of these enzymes have been exploited in vitro to provide many of the protocols currently used in nucleic acid manipulations.
These polymerases often add a single deoxyadenosine, in a templateindependent fashion, to the 3. Compared to newer methods such as gibson assembly, ta cloning is less efficient and nondirectional, and the method cannot be used to assemble more than one fragment at a time. Instructions for use of products a60, a80, a3600, a3610. The pgemt vector systems are convenient systems to clone pcr products. How to clone a pcr amplified product in a pet expression vector. There are a multitude of these and especially since you are cloning using a promega kit, i would suggest using gotaq. Base pair complementarity can orient the pcr product with respect to the plasmid sequence. Introduction the cloning efficient than bluntended ligation for the cloning of pcrof a dna fragment into a plasmid vector is a routine procedure in recombinant dna technology. Download gblocks gene fragments cloning protocols pdf. The ligafast system is bluewhite cloning qualified, providing a higher level of quality control for enzymes used in cloning applications. The pgem t and pgem t easy vectors are linearized vectors with a single. A novel series of highefficiency vectors for ta cloning and.
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