Get cells out of freezer, thaw on ice for 5 minutes 2. Promega corporation is a worldwide leader in applying biochemistry and molecular biology to the development of innovative, highvalue products for the life sciences. If possible, only visualize the pcr product with a longwave uv source. To be the most responsive supplier of biological reagents and reagent systems used in research and applied technology applications worldwide. Promega pgemt and pgemt easy vector systems fisher. Mar 16, 2019 download pgem t and pgem t easy vector systems promega book pdf free download link or read online here in pdf. The pgemt vector systems are convenient systems to clone pcr products generated by certain thermostable polymerases. Thus, several options exist to remove the desired insert dna with a single restriction digestion. To determine if large dna fragments can be cloned successfully into promegas pgemt vectors, a 7. Contains t7 and sp6 rna polymerase promoters flanking a multiple cloning region within the alphapeptide coding region of betagalactosidase. The pgem t and pgem t easy vectorsa,b are linearized vectors with a single 3. A versatile zero background tvector system for gene cloning and.
The pgem dna markers have 15 phenolextracted, ethanolprecipitated dna fragments ranging in size from 36bp to 2,645bp. Ligation reactions using this buffer may be incubated for 1 hour at room temperature. Promega pgemt and pgemt easy vector systems print promega pgemt and pgemt easy vector systems. Troubleshooting experiments automation installation and support global availability multilanguage capabilities toll free in usa. Shop online for a wide selection of promega pgemt and pgemt easy vector systems convenient systems for the routine subcloning of pcr products. The pgem t vector is a highefficiency ta cloning vector which contains multiple cloning sites as shown below. Plasmid isolation was performed using qiaprep spin miniprep kit as per the manufacturers instructions. The pgema,bt and pgemt easy vector systems are convenient systems for the cloning of pcrc products. Jan 08, 2020 one of the easiest methods for cloning bluntended dna fragments including pcr products is tvector cloning, such as with pgemt or pgemt easy vector systems.
An aliquot of 1mg the total rna was reverse transcribed with 50cds primer a and smart ii a oligonucleotide provided in the kit to obtain the 50ready cdna using the smarttm race cdna ampli. Technical manual pgem t and pgem t easy vector systems instructions for use of products a60, a80, a3600 and a3610. The pgem9zf vector is a recombinant plasmid that provides a versatile range of cloning strategies, efficient synthesis of rna in vitro and the production of singlestranded dna. Since it has atoverhang, so will this affect the type of gene. Promega manufacturing and delivery systems continue to be fully operational during the covid19 outbreak. Both the pgemt and pgemt easy vectors contain numerous restriction sites within the multiple cloning region. The pgemt easy vector systems are convenient systems to clone pcr products. Vector database is a digital collection of vector backbones assembled from publications and commercially available sources. These single 3t overhangs at the insertion site greatly improve the efficiency of ligation of a pcr product into the plasmid. Welcome to vector database vector database is a digital collection of vector backbones assembled from publications and commercially available sources.
For preparation of standard curve, lacz and reca gene were cloned individually in pgemt easy vector promega and the constructs were named pjj8 and pjj9 respectively. View notes pgem from chem 1a at san jose state university. Please contact promega technical services if you have questions on use of this system. T vectors are linearized plasmids that have been treated to add 3. All books are in clear copy here, and all files are secure so dont worry about it. Recognition sites for bstz i, ecor i, and not i flank the insertion site several options are available for removal of the desired insert dna with a single restriction digestion.
Incubate at room temperature for 1 hour or overnight at 4c 3. Promega pgemt and pgemt easy vector systems pgem t easy. Download pgem t and pgem t easy vector systems promega book pdf free download link or read online here in pdf. The pgemt easy vector systems are convienent systems to clone pcr products. The pgem t and pgem t easy vectors are linearized vectors with a single 3. May 21, 20 this feature is not available right now. The position of the t is indicated by in the pgemt vector sequence. The pgemt easy vector systems offer all of the advantages of the pgemt vector systems with the added convenience of recognition sites for bstzi, ecori and noti flanking the insertion site. The pgem t 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 pgem t and pgem t easy vector systems are convenient systems for the cloning of pcr products. Briefly centrifuge the pgemt or pgemt easy vector and control insert. These polymerases often add a single deoxyadenosine, in a templateindependent fashion, to the 3.
Promega corporation 2800 woods hollow road madison, wi 537115399 usa. 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 ligafast rapid dna ligation system is designed for the efficient ligation of stickyended dna inserts into plasmid vectors in just 5 minutes bluntended inserts in as little as 15 minutes. Recognition sites for bstz i, ecor i, and not i flank the insertion site several options are available for removal of the desired insert dna with a. The ligation and subsequent transformation of this positive control can give you a lot of information with regard to the ligation and transformation of your insert. There is no genbank accession number for the pgemt easy vector. They offer all of the advantages of the pgemt vector systems with ecori and. Promegas pgemt vector systems are very dependable and versatile tools to routinely clone fragments of dna into reliable plasmids. Nov 10, 2009 promegas pgemt vector systems are very dependable and versatile tools to routinely clone fragments of dna into reliable plasmids. We tested the promega t4 dna ligase, which is included in the pgemt easy.
These unique markers are generated from separate digests of pgem3 vector dna with hinfi, rsai and avaii later combined to form the markers. Quick protocol 1 pgem t and pgem t easy vector systems instructions for use of products a60, a80, a3600 and a3610. The pgem t vector is ready to use in ligation reactions, prepared by cutting the pgem 5zf. A major pgemt easyrelated selection system drawback is the growth of falsepositive colonies white colonies without the insert and falsenegative colonies blue ones with the insert recombined. The pgemt vector is a highefficiency ta cloning vector which contains multiple cloning sites as shown below. The pgemt and pgemt easy plasmid vectors are essentially the same but with one important difference. They offer all of the advantages of the pgemt vector systems with added convieneice of recognition sites for ecori and noti flankin the insertion site. The pgemt and pgemt easy plasmid vectors are essentially the same but with one.
This is a free resource for the scientific community that is compiled by addgene this page is informational only this vector is not available from addgene please contact the manufacturer for further details. Double digestion may also be used to release the insert from the vector. This method takes advantage of the a overhang added by a pcr enzyme like taq dna polymerase. Protocol for ligations using the pgem t and pgem t easy vectors and the 2x rapid ligation buffer 3.
This fragment was purified using the wizard pcr preps dna purification system, and cloned into the pgemt easy vector using a vector. The plasmid contains t7 and sp6 rna polymerase promoters flanking a multiple cloning region within the. Bluntend dna fragments can be ligated into promegas tvectors if they are first tailed using datp and taq dna polymerasea 1. T overhangs to match the a overhangs of the insert. Each promega pcr cloning system is provided with a control insert. The pgem t and pgem t easy vector systems are supplied with 2x rapid ligation buffer. We tested the promega t4 dna ligase, which is included in the pgemt easy vector system, and the t4 dna ligase from usb corporation. Equipment necessary 10 l micropipette 200 l micropipette l micropipette 37 c incubator 37 c shaking incubator 42 c heat block.
Rapid ligation is based on the combination of t4 dna ligase with a unique 2x rapid ligation buffer. The toverhangs at the insertion site greatly improve the efficiency of. There is no genbank accession number for the pgem t easy vector. T overhangs at the insertion site greatly improve the efficiency. 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. 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 pgem t easy vector systems offer all of the advantages of the pgem t vector systems with the added convenience of recognition sites for bstzi, ecori and noti flanking the insertion site.
The ligations using promega t4 dna ligase consistently gave a very high cloning efficiency. How to multiply pgem t easy vector in the laboratory. We have generated a multifunctional vector, pgemwiz, to assemble inverted repeats targeting any drosophila genes by shuttling intron 2 of the drosophila gene white from pwiz into a cloning vector, pgems1 fig. Multiple cloning site is flanked by recognition sites for the restriction enzyme bstz i singleenzyme digestion allows release of the insert. The pgemt and pgemt easy vectorsa,b are linearized vectors with a single 3. Feb 14, 2009 we tested the promega t4 dna ligase, which is included in the pgem t easy vector system, and the t4 dna ligase from usb corporation. As pgems1, pgemwiz uses pgemt promega as its backbone. The parent vector is linearized at the position indicated by in this pgemt easy vector sequence and a t is added at each end. Productpgemt easy vector system i product codea60 substance number description amount symbols a7 pgemt easy 1 a363 control insert dna 1 m180 t4 dna ligase 1 c671 2x rapid ligation buffer, t4 dna ligase 1. Cloning and molecular characterization of hbcoi1 from. Instructions for use of products a60, a80, a3600, a3610. Briefly centrifuge the pgem t or pgem t easy vector and control insert dna tubes to collect contents at the bottom of the tube.
Description the pgem t and pgem t easy vector systemsa,b are convenient systems for the cloning of pcr products. Pgem t and pgem t easy vector systems promega pdf book. May i know if it is true that gene with any sequence also can be inserted into pgemt easy. Technical manual pgemt and pgemt easy vector systems instructions for use of. Read online pgem t and pgem t easy vector systems promega book pdf free download link book now. May i know if it is true that gene with any sequence also can be inserted into pgem t easy. Briefly centrifuge the pgem t or pgem t easy vector and control insert dna tubes to collect the contents at the bottom of the tubes. The incubation period may be extended to increase the number of colonies after transformation. Strong synthetic stationary phase promoterbased gene. This is a free resource for the scientific community that is compiled by addgene.
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