The most basic need in pcr subcloning is a simple, general cloning vector. May 21, 20 this feature is not available right now. Promega pgemt and pgemt easy vector systems fisher. Description the pgem t and pgem t easy vector systemsa,b are convenient systems for the cloning of pcr products. The pgem t vector is a highefficiency ta cloning vector which contains multiple cloning sites as shown below. This is a free resource for the scientific community that is compiled by addgene. Followed the ligation protocol as per promega instuctions. The pgem t easy vector system ii contains jm109 competent cells in addition to all of the pgem t easy vector system i components.
This sounds dumb and i think i know the answer but looking at the vector map has me confused. T overhangs at the insertion site greatly improve the efficiency. The vector allows preparation of singlestranded dna due to its f1 origin of replication. What is the difference between the agarose gel views of. Yes you can insert any gene sequence in pgemt easy vector but one thing you.
The pgem t and pgem t easy vector systems are convenient systems for the cloning of pcr products. Transformation protocol when using the pgemt and pgemt easy vector ligation reactions 1. 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. Equilibrate the plates to room temperature 37 c prior to plating. Double digestion may also be used to release the insert from the vector. The pgem t and pgem t easy vector systemsa,b are convenient systems for the cloning of pcr products. 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. These single 3t overhangs at the insertion site greatly improve the efficiency of ligation of a pcr product into the plasmid. 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. This allows the insert dna to be removed with a single restriction digest using either of these enzymes.
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. Rob partons lab contains the insert rab5c and is published in cell logist. The pgemt easy vector systems are convenient systems for cloning pcr products. Home resources plasmid files basic cloning vectors pgemt easy. The pgem t and pgem t easy vector systems are supplied with 2x rapid ligation buffer. I have been trying to subclone an insert having 2300 bp into pgem t easy vector. Overview protocols specifications resources pcr cloning with bluewhite selection and easy insert excision the pgemt easy vector systems are. The pgem t and pgem t easy vectorsa,b are linearized vectors with a single 3.
The pgem t vector cloning region is flanked by recognition sites for the enzyme bstzi. Incubate at room temperature for 1 hour or overnight at 4c 3. The pgemt and pgemt easy vector systemsa,b facilitate the cloning of pcrc products by providing linear vectors that have a single thymidine extension. Since it has atoverhang, so will this affect the type of gene. The pgemt easy vector systems are convenient systems for cloning pcr. May i know if it is true that gene with any sequence also can be inserted into pgemt easy. The pgem t vector systems are convenient systems to clone pcr products generated by certain thermostable polymerases. There is no genbank accession number for the pgem t easy vector. The pgem r t easy vector has been linearized with ecorv at base 60 of this sequence indicated by an.
It is very obvious that by running the single digested plasmids side by side you can find out the difference with t. Prepare 1 lbampicillinxgal lba plates are 1ml of 100mm amp in a liter, 32 ul of 50 ugul xgal per plate plates for each ligation reaction. Transformation protocol when using the pgem t and pgem t easy vector ligation reactions 1. Learn about the latest plasmid technologies and research tools. They offer all of the advantages of the pgem t vector systems with ecori and noti sites flanking the insertion site. The pgemt easy vector multiple cloning region is flanked by recognition sites for the restriction enzymes ecori, bstzi and noti, providing three singleenzyme. Thus several options for removal of the desired insert dna with a single restriction. Technical manual pgem t and pgem t easy vector systems instructions for use of products a60, a80, a3600 and a3610. The pgemt vector systems are convenient ta vector systems for quickly cloning. May i know if it is true that gene with any sequence also can be inserted into pgem t easy.
The toverhangs at the insertion site greatly improve the efficiency of. These polymerases often add a single deoxyadenosine, in a templateindependent fashion, to the 3. The insertion site is flanked by bstzi, ecori, and noti sites. The pgem t easy vector systems are convenient systems for cloning pcr products.
Transformation protocol when using the pgemt and pgemt easy. T%20and%20pgemt%20easy%20vector%20systems%20protocol. Have questions about your order, deposit, or a plasmid. Alternatively, a doubledigestion may be used to release. The pgemt vector is a highefficiency ta cloning vector which contains multiple cloning sites as shown below. The pgemt easy vector multiple cloning region is flanked by recognition sites for the restriction enzymes ecori, bstzi and. Promega pgemt and pgemt easy vector systems pgem t easy. Alternatively, a doubledigestion may be used to release the insert from either vector. Mar 11, 2020 we have demonstrated that gotaq long pcr master mix successfully generated dna fragments that could be ligated into pgem t easy vector system without an atailing procedure, and with ligation efficiencies similar to those observed with the gotaq green master mix. Transformation protocol when using the pgemt and pgemt. A versatile zero background tvector system for gene. The pgem t easy vector systems are convenient systems to clone pcr products. Comparing cloning efficiency of the pgemt and pgemt easy. Receive the latest news, hot plasmids, discounts and more.
Pcr products can be easily cloned into a linearized vector with 3. 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. The parent vector is linearized at the position indicated by in this pgem t easy vector sequence and a t is added at each end. Get cells out of freezer, thaw on ice for 5 minutes 2. The pgem t and pgem t easy vector systems gave a high number of recombinants across a broad range of insert sizes 0. For pgem t easy vector, you can use simple primer for cloning and you must to use taq dna polymerase for gene amplification. To see this sequence with restriction sites, features, and translations.
Our teams are in regular contact with suppliers and distributors worldwide and are taking all steps necessary to address both demands for diagnostic tools and reliable delivery of all products as quickly as possible. The pgemt easy vector systems are convenient systems to clone pcr products. Becuase taq is able to add a to 3 overhang of pcr product. As far i understand with your question, that you want to know the difference between the vector and cloned plasmids on agarose gel. I need to know what are the differences between both pgemt and pgemt easy vector. How to multiply pgem t easy vector in the laboratory. The pgem t easy vector systems are convenient systems to clone pcr products generated by certain thermostable polymerases. A versatile zero background tvector system for gene cloning and.
We evaluated the cloning efficiency of different size pcr products into three tvector cloning systems. View notes pgem from chem 1a at san jose state university. The pgem t and pgem t easy vector systems are designed for just that purpose. This page is informational only this vector is not available from addgene please contact the manufacturer for further details. 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. Multiple cloning site is flanked by recognition sites for the restriction enzyme bstz i singleenzyme digestion allows release of the insert. Briefly centrifuge the pgem t or pgem t easy vector and control insert dna tubes to collect contents at the bottom of the tube. They offer all of the advantages of the pgem t vector systems with the added convenience of recognition sites for ecori and noti flanking the insertion site. They offer all of the advantages of the pgemt vector systems with ecori and noti sites flanking the insertion site. The primers i used have snabl and notl site in forward and reverse primer end respectively.
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