Us9822348b2 us15316,232 us201515316232a us9822348b2 us 9822348 b2 us9822348 b2 us 9822348b2 us 201515316232 a us201515316232 a us 201515316232a us 9822348 b2 us9822348 b2 us 9822348b2 authority us united states prior art keywords expression t7 plasmid expression system rna polymerase prior art date 20140701 legal status the legal. And as it happens, cloning strategies that work for other types of dna fragments dont work at. The j b c 2003 by the american society for biochemistry. A simple and efficient method for assembling tale protein based on plasmid library zhiqiang zhang. Highcopynumber pgem t easy vector contains t7 and sp6 rna polymerase promoters. Once the primers are synthesized, we aliquot them for the dna sequencing and save the rest in the mc easy depositoryfor possible future usage from our customers. Highthroughput phenotyping of avoidance learning in mice. Description the pgem t and pgem t easy vector systemsa,b are convenient systems for the cloning of pcr products. Dna tubes to collect contents at the bottom of the tube. The high copy number pgem t and pgem t easy vectors contain t7 and sp6 rna polymerase promoters flanking a multiple cloning region within the alphapeptide coding region of the enzyme betagalactosidase. The integrative promoter probe vector ptpro1 contains a promoterless kanamycinresistance gene to examine the expression of genes as a single copy. Three microliter was ligated in pgemt easy cloning kit promega, fitchburg, wi, usa, transformed into. Three clones of each cloned pcr product were sequenced by the method described by sanger et al, using the t7 primer and the big dye terminator cycle sequencing ready reaction kit pe applied biosystems, foster city, ca, usa. The nucleotide sequence of the insert in the resulting pgemtcatc vector was confirmed by dna sequencing using the t7 and sp6 promoter sequencing primers.
Design an external forward primer that includes at its 3. Indian journal of biotechnology vol 12, october 20, pp 489492 development of scar based molecular markers for identification of different species of azolla g abraham1, neha pandey2, vagish mishra3, anis ahmad chaudhary4, altaf ahmad4, rajendra singh5 and p k singh6 1centre for conservation and utilization of blue green algae, 3national research centre for plant. Competitive pcr for precise nucleic acid quantification. The high copy number pgemt and pgemt easy vectors contain t7 and sp6 rna. Supporting information boosting crisprcas9 multiplex. If initial experiments with your pcr product are suboptimal, ratio optimization may be necessary. The pgem t and pgem t easy vectors are approximately 3kb and are supplied at 50ngl. A, schematic structures of the rnai plasmids used in this study. The primers i used have snabl and notl site in forward and reverse primer end respectively. Kanamycin resistance an overview sciencedirect topics. This allows the insert dna to be removed with a single restriction digest using either of these enzymes. The highcopynumber pgem t and pgem t easy vectors contain t7 and sp6 rna polymerase promoters flanking a multiple cloning region within the. Learn about the latest plasmid technologies and research tools. For quantitative rtpcr, the dnase i treated total rnas were reversetranscribed to produce cdnas using a specific primer grnar along with mumlv new england biolabs according to the manufacturers instructions.
Briefly centrifuge the pgemt or pgemt easy vector and control insert. The only difference between pgemt and pgemt easy is in the multiple cloning site mcs. The dna was amplified by pcr in 50l volumes with 50pmol of each primer. Instructions for use of products a60, a80, a3600, a3610. April, 1998pcr based cloning kits table the end table pdf format pcr has found applications in almost every imaginable facet of molecular biology, and for many applications, looking at a band on a gel is not enough. Primers for amplification of nitrous oxide reductase genes. 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. How to multiply pgem t easy vector in the laboratory. The pgemt easy vector systems are convenient systems for cloning pcr. The toverhangs at the insertion site greatly improve the efficiency of. The pgemt vector is a highefficiency ta cloning vector which contains multiple cloning sites as shown below. Sixteen white colonies were selected and resuspended in 15 ml of lb media supplemented with 25 mgl ampicillin, 1 ml of the mutants were ampli.
Pcr purified fragments were cloned into pgemt easy vector promega corporation. The 1218 version of this technical manual was revised to remove references to discontinued products in the notes on sequencing primers in section 5. For pgem t easy vector, you can use simple primer for cloning and you must to use taq dna polymerase for gene amplification. Sequencing of the positive clones were done by sanger method using 96 capillary high through put sequencer. S1 available with the online version of this paper. A simple and efficient method for assembling tale protein.
To generate a standard curve for nosz1fnosz1r and nosz2fnosz2r, the pcr product from p. Promega corporation2800 woods hollow road madison, wi 537115399 usa toll free in usa 8003569526 telephone 6082744330 fax 6082772601 printed in usa. You may consider to ask the plasmid from the group to try out in your lab. Sequencing, expressing, mutatingall require cloning. To calculate the appropriate amount of pcr product insert to include in the ligation reaction, use the following. Receive the latest news, hot plasmids, discounts and more. Updated to allow for in vitro transcription with both sp6 and t7 promoters. The high copy number pgem t and pgem t easy vectors contain t7 and sp6 rna polymerase promoters flanking a multiple cloning site mcs within the. The pcr product 2 ml was ta cloned into pgemt easy vector promega following the manufacturers protocol. Abi 3,730 xl xcelris, india with t7 and sp6 universal primer.
Melting curve analysis supported the specificity of the qpcr primers see fig. Slp4agranuphilina inhibits densecore vesicle exocytosis through interaction with the gdpbound form of rab27a in pc12 cells received for publication, december 23, 2002, and in revised form, january 21, 2003 published, jbc papers in press, february 17, 2003, doi 10. Decreases in the amounts of oma1 and oma2 trasncripts caused by feeding rnai detected by quantitative rtpcr. T%20and%20pgemt%20easy%20vector%20systems%20protocol. T7 and sp6 promoter primers were applied on pgemt easy derived vectors while t7. Pgemt is a linearized cloning vector that can not be multiplied. The vectors provide convenient t7 and sp6 promoters that can serve as binding sites for sequencing primers, or for promoting in vitro transcription on either strand of the insert when the appropriate rna polymerase is used. The promoter and multiple cloning sequence of the pgemt vector. Colonies were suspended in 50l sterile water, boiled for 10 minutes, centrifuged at 16,000. Becuase taq is able to add a to 3 overhang of pcr product. Dna cloning is an essential tool regarding dna recombinant technology as it allows the replication of foreign dna fragments within a cell. Have questions about your order, deposit, or a plasmid. Easy cloning of all pcr products, including blunt and ta ends. T7 and sp6 rna polymerase promoters flank a multiple cloning region within the.
Riboprobe synthesis for in situ hybridization martindale lab. Results and discussion blastn seach of endoglucanase gene of b. The top strand corresponds to the rna synthesized by t7 rna polymerase. The size of the pcr product has a limitation of cloning into pgemteasy vectors. Takeda using primers designed to amplify a 150base pair bp fragment within the tbox ruvinsky et al. There is no genbank accession number for the pgem t easy vector. An altered method of feeding rnai that knocks down. Bsaisite removed from the ampicillinresistant gene allows for the cloning of golden gate assembly modules.
A 150 bp product was cloned into pgemt easy promega, madison, wi, labeled with 32p and used as a probe to screen the ovary library. Each structure indicates the location of inserted cdnas oma1, oma2, daf16, and gfp in the multiplecloning site of. Insertional inactivation of the alphapeptide allows recombinant clones to be directly identified by bluewhite screening on indicator plates. P3 plxsn plxsnf plxsnr pmalc2e male primerj46583 mf47. Cloning, expression, and activity analysis of human. In situ hybridization for tilv food and agriculture.
In vitro transcription with digutp using t7 rna polymerase. Briefly centrifuge the pgem t or pgem t easy vector and control insert. I need to know what are the differences between both pgemt and pgemt easy vector. The only difference between pgemt and pgemt easy is in the. So that the next time when you order the sequencing using the same primers, you will save time and money by choosing the primers we saved for you in the mc easy depository. 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 cdna fragments of syt i c2a mutants with a t7 tag were then excised from the pgemt easy vector by noti digestion and subcloned into the noti site of a modified pefbos mammalian expression vector 59, 60. Yes you can insert any gene sequence in pgemt easy vector but one thing you.
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