I need to know what are the differences between both pgemt and pgemt easy vector. The pgem t easy vector systems are convenient systems to clone pcr products generated by certain thermostable polymerases. Pcr cloning with bluewhite selection and easy insert excision. Comparing cloning efficiency of the pgem t and pgem t easy vectors to the topo ta cloning vectors. The bottom strand corresponds to the rna synthe sized by sp6 rna polymerase. We evaluated the cloning efficiency of different size pcr products into three tvector cloning systems. The pgem t vector is a highefficiency ta cloning vector which contains multiple cloning sites as shown below. By continuing to use this site, you agree to the use of cookies. Alternatively, a doubledigestion may be used to release the insert from. Previous publications have described the use of restriction enzyme xcmi kovalic et al. The insertion site is flanked by bstzi, ecori, and noti sites. Briefly centrifuge the pgemt or pgemt easy vector and control 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. Multiple cloning site is flanked by recognition sites for the restriction enzyme bstz i singleenzyme digestion allows release of the insert.
The pgemt easy vector systems are convenient systems to clone pcr products generated by certain thermostable polymerases. How to multiply pgem t easy vector in the laboratory. Molecular cloning is a process of isolation of a specific dna fragment and transfer of this fragment into a plasmid vector. 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. The pgem t and pgem t easy vectors a,b are linearized vectors with a single 3. Yes you can insert any gene sequence in pgemt easy vector but one thing you. Aversatile zero background tvector system for gene cloning. The linearized ptg19t vector with 3dt overhangs prevent vector recircularization, therefore resulting in high percentage of recombinant clones and low background. May 21, 20 this feature is not available right now. Promega pgemt and pgemt easy vector systems pgem t easy. Ordering information the number of white coloniesng vector dna 400 and 2,400bp. The most basic need in pcr subcloning is a simple, general cloning vector. The pgem t and pgem t easy vector systems gave a high number of recombinants across a broad range of insert sizes 0.
A versatile zero background tvector system for gene cloning. The pgemt vector cloning region is flanked by recognition sites for the enzyme. As you are presumably cloning by pcr, normal taq based pcr results in a single bp a overhang on the end of your sequence, this can be used to clone into t vectors such as pgem t where the vector comes precut with a t overhang and dna ligase conjugated to the ends, which makes cloning quick and easy. The pgem t and pgem t easy vector systemsa,b are convenient systems for the cloning of pcr products. 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.
Since it has atoverhang, so will this affect the type of gene. The promoter and multiple cloning sequence of the pgem t panel a and pgem t easy panel b vectors. Mar 11, 2020 using gotaq long pcr master mix for tvector cloning. This website uses cookies to ensure you get the best experience. The top strand of the sequence shown corresponds to the rna synthesized by t7 rna polymerase. Description the ptg19t vector is designed for rapid and efficient cloning of pcr products with 3da overhangs. This allows the insert dna to be removed with a single restriction digest using either of these enzymes. In addition, the cloning process can be quicker than the pgem system. The pgem t and pgem t easy vector systems are designed for just that purpose. Cloning bluntend dna fragments into the pgem t vector systems by gary kobs promega corporation bluntend dna fragments can be ligated into promegas tvectors if they are first tailed using datp and taq dna polymerasea 1.
The pgem t easy vector multiple cloning region is flanked by recognition sites for the restriction enzymes ecori, bstzi and noti, providing three singleenzyme digestions for release of the insert. These polymerases often add a single deoxyadenosine, in a templateindependent fashion, t. The pgemt easy vector multiple cloning region is flanked by recognition sites for the restriction enzymes ecori, bstzi and. May i know if it is true that gene with any sequence also can be inserted into pgemt easy. Equipment necessary 10 l micropipette 200 l micropipette l micropipette 37 c incubator 37 c shaking incubator 42 c heat block. The pgem t vector systems are convenient systems to clone pcr products generated by certain thermostable polymerases. I find that it is more efficient than pgem t easy and that i get more white colonies with my insert. The pgemt easy vector multiple cloning region is flanked by recognition sites for the restriction enzymes ecori, bstzi and noti, providing three singleenzyme digestions for release of the insert. T overhangs at the insertion site greatly improve the efficiency. Vortex the 2x rapid ligation buffer vigorously before each use.
Get cells out of freezer, thaw on ice for 5 minutes 2. Comparing cloning efficiency of the pgemt and pgemt easy. The atailed product can be added directly to the ligation as described in the pgem t or pgem t easy vector protocol. Home resources plasmid files basic cloning vectors peasyt3 linearized peasyt3 linearized ta cloning vector that allows the cloned pcr product to be excised with ecori or noti. These polymerases often add a single deoxyadenosine, in a templateindependent fashion, to the 3. A cloning vector is a small piece of dna that can be stably maintained in an organism, and into which a foreign dna fragment can be inserted for cloning purposes. A versatile zero background tvector system for gene cloning and. The pgem t easy vector systems are convenient systems to clone pcr products. In this article, the results of two different atailing protocols were evaluated using a pgem tb,c easy vector. Feb 14, 2009 although the former has been used to produce commercial cloning kits like the pgem t system, we selected the restriction endonuclease digestionmediated strategy to develop a ta cloning vector system because this approach is easy to use for individual laboratories. The only difference between pgem t and pgem t easy is in the multiple cloning site mcs. Overview protocols specifications resources pcr cloning with bluewhite selection and easy insert excision the pgem t easy vector systems are convenient systems to clone pcr products generated by certain thermostable polymerases. Description the pgem t and pgem t easy vector systemsa,b are convenient systems for the cloning of pcr products.
Do these polishing components present in the master mix affect my cloning efficiency if my insert already has blunt ends. Alternatively, a doubledigestion may be used to release the insert from either vector. How can the cloning vector work with both bluntended amplicons and singlebase overhangcontaining amplicons. 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.
Double digestion may also be used to release the insert from the vector. The pgem t and pgem t easy vector systems are supplied with 2x rapid ligation buffer. The pgem t vector cloning region is flanked by recognition sites for the enzyme bstzi. The pgem t easy vector systems are convenient systems for cloning pcr products. The pgem t and pgem t easy vector systems are convenient systems for the cloning of pcr products. Pcr products can be easily cloned into a linearized vector with 3. Incubate at room temperature for 1 hour or overnight at 4c 3. There is no genbank accession number for the pgem t easy vector. They offer all of the advantages of the pgem t vector systems with ecori and noti sites flanking the insertion site. The mcs of the pgemt easy vector contains sequences on either side of the insert that are recognized by.