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            pUCm-T載體說明書

            發布日期:2017-01-07

            R04002    40T         R04004   40T

            R04003    100T        R04005   200T

            Components

            COMPOSITIONS

            R04002

            R04003

            R04004

            R04005

            pUCm-T Vector

            2μg

            10μg

            1μg

            10μg

            10×Ligation Buffer

             

             

            100μl

            400μl

            50% PEG4000

             

             

            100μl

            400μl

            T4 Ligase,5U/μl

             

             

            200U

            1000U

            Sterilized ddH2O

             

             

            1ml

            1ml

            Intoduction

            The T-Vector PCR Product Cloning Kit is suitable for cloning of PCR products with additional A at 3’ end.The special ligation system provided by the kit enables customs to finish ligation in 1-2 hours.

            pUCm-T vector of our company is design for simplifying cloning of PCR products.Many thermal stable DNA polymerase, PCR products amplified by DNA polymerase such as Taq, Tth DNA produce additional A at 3’ edn which could be easily ligated to T vector with additional T.    

            pUCm-T of our company is a kind of novel pUC derivative T vector, the multiple restrict sits with most of them single site and adjusted β-galactose reading frame make it easily for screening target clone through blue and white plaque.Specially designed two Pst I sites beside inserted fragments make it easy for screening target clone by Pst I digestion, at same time inserted framents also could be screened by cheap and efficient restrict enzymes such as EcoR I and Hind III double digestion.Inserted fragments also could be sequenced using universal primers M13 and T7 promoter primer.In vitro transcription could be processed through site of T7 RNA polymerase promoter in pUCm-T.

            Technical materials of our product are displayed at the end of protocol,the complete sequence of our T vector are same as pUC19GenBank Accession Number M77789except difference at multiple cloning site.

            Preparation of ligation reaction:

            Purification of PCR products or not depends on quality of amplified prduct. If PCR products are very specific,purification of PCR products is not necessary,But if plasmids were used as template,it is necessary to purify PCR product,because plasmid template could form white colony after transformation.PCR products could be separated by agarose electrophoresis.

            PCR products amplified by TaqTthAmpliTaqKlenTaq DNA polymerase bear additional A at 3’end.Taq DNA polymerase co-amplify with PfuPwoTli or Deep vent DNA polymerase wich posses 3’—5’ exonuclease activity may bear a additional A at 3’end. PCR products posses additional A at 3’end could be ligated to pUCm-T. PCR products amplified by DNA polymerase with 3’—5’ exonuclease activity is blunt end,cloning of this kind of fragments need to add additional A to blunt end.

            MAP OF pUCm-T VECTOR 

            pUCm-T|T載體|質粒

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