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      酵母雙雜交系統(tǒng)EasyClone normalized cDNA library construct

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      酵母雙雜交系統(tǒng)EasyClone normalized cDNA library construction package
      Fast and Easy Synthesis of Highly Complex cDNAs
      Directional cloning of cDNAs
      酵母雙雜交系統(tǒng)EasyClone normalized cDNA library construct 產(chǎn)品信息

       

      酵母雙雜交系統(tǒng)EasyClone normalized cDNA library construction package
      Fast and Easy Synthesis of Highly Complex cDNAs
      Directional cloning of cDNAs
      The EasyClone Normalized cDNA Library Construction Kit is designed to synthesize full-length-enriched
      double stranded (ds) cDNA from total or polyA+ RNA, followed by normalization of amplified cDNA and subcloning into an appropriate library vector. The kit is based on a novel technology utilizing the specific features of MMLV-based reverse transcriptase to create full length enriched double stranded cDNA from total or polyA+ RNA samples. cDNAs are amplified by representational PCR and cloned directionally into library vectors for use with yeast two-hybrid systems such as Dualsystems' DUALmembrane, DUALhybrid, and DUALhunter protein interaction screening kits.
       
       
      No extensive know-how in library construction is needed - the kit supplies all reagents needed for library construction and the detailed manual will guide you through all steps of construction.
       
      Construction of normalized cDNA libraries
      Normalization is the process of removing abundant RNAs from the starting material. Normalized cDNA libraries contain approximay equal amounts of all RNAs and therefore increase the occurrence of rare proteins. This significantly increases the quality of yeast two-hybrid screens.
       
      Figure 1: qPCR of normalized and not normalized human spleen cDNA. For Actin, a decrease
      of 75.8% was reached. For hGAPDH, the decrease was 60.6%. The amount of these two high
      abundant cDNAs was lowered, increasing the quality of the cDNA library.
       
       
      KIT CONTENTS - Box 1
      5X first strand buffer
      80 µl
      DTT (20 mM)
      30 µl
      10X dNTP mix (10 mM each)
      110 µl
      PlugOligo-3M adapter (15 µM)
      25 µl
      CDS-3M adapter (10 µM) 
      25 µl
      EasyClone reverse transcriptase
      20 µl
      IP-solution
      130 µl
      Control RNA template (0.5 µg/µl)
      15 µl
      Sterile RNase free water
      1.8 ml
       
      KIT CONTENTS - Box 2
      50X EasyClone polymerase mix   
      50 µl
      10X EasyClone buffer
      300 µl
      PCR Primer M1 (10 µM)
      170 µl
      Sterile RNase free water
      1.8 ml
      Control amplified cDNA sample 1
      25 µl
      Control amplified cDNA sample 2
      25 µl
       
      KIT CONTENTS - Box 3
      NORMALIZER enzyme, lyophilized
      50 units
      NORMALIZER storage buffer (50 mM Tris-HCl pH 8.0)
      70 µl
      4x Hybridization buffer (200 mM Hepes, pH 7.5, 2 M NaCl)
      70 µl
      2x NORMALIZER master buffer (100 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 2 mM DTT)
      250 µl
      NORMALIZER stop solution (5 mM EDTA)
      500 µl
      NORMALIZER control template, 100ng/μl
      20 µl
      ACTB plasmid (100 ng/μl)
      50 µl
      UBC plasmid (100 ng/μl)
      50 µl


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