Is the insert size too big for a pgem t easy vector. The pgem t and pgem t easy vectorsa,b are linearized vectors with a single 3. The toverhangs at the insertion site greatly improve the efficiency of ligation of pcr products by preventing recircularization of the vector and providing a compatible overhang for pcr products. There is no genbank accession number for the pgemt easy vector.
Alternatively, a doubledigestion may be used to release the insert from either vector. I find that it is more efficient than pgem t easy and that i get more white colonies with my insert. These polymerases often add a single deoxyadenosine, in a templateindependent fashion, to the 3. The mcs of the pgemt easy vector contains sequences on either side of the insert that. The pgem t vector is a highefficiency ta cloning vector which contains multiple cloning sites as shown below. The linearized ptg19t vector with 3dt overhangs prevent vector recircularization, therefore resulting in high percentage of recombinant clones and low background. Description the pgem t and pgem t easy vector systemsa,b are convenient systems for the cloning of pcr products. The pgem t easy vector systems are convenient systems to clone pcr products generated by certain thermostable polymerases. To be the most responsive supplier of biological reagents and reagent systems used in research and applied technology applications worldwide. Briefly centrifuge the pgemt or pgemt easy vector and control insert. Get cells out of freezer, thaw on ice for 5 minutes 2. Receive the latest news, hot plasmids, discounts and more. I decided to use pgem t easy for doing ta cloning before i obtain a properly digested pcr product.
Incubate at room temperature for 1 hour or overnight at 4c 3. The parent vector is linearized at the position indicated by in this pgemt easy vector sequence and a t is added at each end. Comparing cloning efficiency of the pgemt and pgemt easy. The mcs of the pgem t easy vector contains sequences on either side of the insert that are recognized by the restriction enzymes not i and ecor i. The primers i used have snabl and notl site in forward and reverse primer end respectively. Yes you can insert any gene sequence in pgemt easy vector but one thing you. May 21, 20 this feature is not available right now. The pgemt vector is a highefficiency ta cloning vector which contains multiple cloning sites as shown below. Our teams are in regular contact with suppliers and distributors worldwide to manage inventory of raw materials to ensure continued availability. The pgem t and pgem t easy vector systems are supplied with 2x rapid ligation buffer. These have been validated for stability and high expression. Name vector type resistance marker bacterial resistance source.
Double digestion may also be used to release the insert from the vector. Linearized vector with 3t overhangs and multiple flanking restriction sites for ta cloning of pcr products. The pgemrt easy vector has been linearized with ecorv at base 60 of this sequence indicated by an asterisk and a t added to both 3 ends. Before being able to use the pgem t easy vector does it need to be digested so its linearized or is the insert site already open. Becuase taq is able to add a to 3 overhang of pcr product. The easyclonemarkerfree vector set is a complete kit for the introduction of genes into 11 predetermined easyclone chomosomal loci. Digital collection of empty plasmid backbones from publications and commercially available sources. I find that it is more efficient than pgemt easy and that i get more white colonies with my insert. The map, notes, and annotations on this page and in the sequence map file are ed material.
The promoter and multiple cloning sequence of the pgemt vector. Each of these sites is located between essential genes and is. Sca i 1875 ori pgem t vector 3003bp ampr apa i aat ii sph i bstz i nco i sac ii spe i not i bstz i pst i sal i nde i sac i bstx i nsi i t7 xmn i 1994 nae i 2695 lacz f1 ori 1 start 14 20 26 31 37 46 55 62 62 73 75 82 94 103 112 126 sp6 t t sca i 1890 ori pgem t easy vector 3018bp. Can anybody kindly give details about pgemhe vector map. Promega manufacturing and delivery systems continue to be fully operational during the covid19 outbreak. Quick protocol 1 pgem t and pgem t easy vector systems instructions for use of products a60, a80, a3600 and a3610. Technical manual pgem t and pgem t easy vector systems instructions for use of products a60, a80, a3600 and a3610. The mcs of the pgemt easy vector contains sequences on either side of the insert that are recognized by the restriction enzymes not i and ecor i. The pgem t vector cloning region is flanked by recognition sites for the enzyme bstzi.
Peter hegemanns lab contains the insert humanized photoactivated adenylyl cyclase and is published in j biol chem. The map, notes, and annotations on this page and in the sequencemap file are ed material. Our teams are in regular contact with suppliers and distributors worldwide to manage inventory of raw materials to ensure uninterrupted availability. Briefly centrifuge the pgem t or pgem t easy vector and control insert dna tubes to collect contents at the bottom of the tube. Multiple cloning site is flanked by recognition sites for the restriction enzyme bstz i singleenzyme digestion allows release of the insert. The pgemt vector cloning region is flanked by recognition sites for the enzyme bstzi. The toverhangs at the insertion site greatly improve the efficiency of. The only difference between pgem t and pgem t easy is in the multiple cloning site mcs. View notes pgem from chem 1a at san jose state university. We evaluated the cloning efficiency of different size pcr products into three tvector cloning systems. The pgemt and pgemt easy vector systemsa,b are convenient systems for the cloning of pcr products.
The insertion site is flanked by bstzi, ecori, and noti sites. Before being able to use the pgem teasy vector does it need to be digested so its linearized or is the insert site already open. May i know if it is true that gene with any sequence also can be inserted into pgemt easy. Description the ptg19t vector is designed for rapid and efficient cloning of pcr products with 3da overhangs. I have been trying to subclone an insert having 2300 bp into pgem t easy vector.
May i know if it is true that gene with any sequence also can be inserted into pgem t easy. The pgemt and pgemt easy vectorsa,b are linearized vectors with a single 3. A versatile zero background tvector system for gene cloning and. Since it has atoverhang, so will this affect the type of gene.
Pcr products can be easily cloned into a linearized vector with 3. How to multiply pgem t easy vector in the laboratory. The top strand corresponds to the rna synthesized by t7 rna polymerase. The pgem t and pgem t easy vector systems are convenient systems for the cloning of pcr products. Reagents and solutions solution composition 10x tbe 0. The aim of molecular cloning is to insert the gene of interest into a plasmid vector, which is then inserted into a cell that will express the protein encoded by the gene of interest. For pgem t easy vector, you can use simple primer for cloning and you must to use taq dna polymerase for gene amplification. A single digest with gsz cat will raease inserts c.
Using the topoisomerase enzyme as opposed to dna ligase is meant to be quicker and more efficient. T overhangs at the insertion site greatly improve the efficiency. The pgemt easy vector systems are convenient systems for cloning pcr products. The pgemt and pgemt easy vector systemsa,b facilitate the cloning of pcrc. Promega corporation is a worldwide leader in applying biochemistry and molecular biology to the development of innovative, highvalue products for the life sciences. This allows the insert dna to be removed with a single restriction digest using either of these enzymes. Purple boxes represent the coding sequence of the thrombin cleavage site. This sounds dumb and i think i know the answer but looking at the vector map has me confused. The pgemt easy vector systems are convenient systems to clone pcr products. They offer all of the advantages of the pgemt vector systems with the added convenience of recognition sites for ecori and noti flanking the insertion site. The pgemt and pgemt easy vector systems gave a high number of recombinants across a broad range of insert sizes 0. The pgemt easy vector systems are convenient systems to clone pcr products generated by certain thermostable polymerases.
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