Second Strand cDNA Synthesis Kit-dNTP based

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Second Strand cDNA Synthesis Kit-dNTP based
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Cat.No.: G475
Quantity: 25 x 50 µl reactions
Stock: In Stock
Price: $360.00
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Data Sheet
Print Version
Description Second Strand cDNA Synthesis Kit is an efficient system of generating double stranded cDNA from first strand cDNA as a template. The E. coli RNase H nicks the RNA in the DNA:RNA hybrid, while the E. coli DNA Polymerase replaces the RNA with deoxyribonucleotides. The E. coli DNA Ligase completes the double stranded DNA formation by linking the gaps between the newly synthesized cDNA strand. The dNTP based kit (Cat. No. G475) and dNTP/dUTP based kit (Cat. No. G476) provides different combination of deoxyribonucleotides tailored to the end user’s needs and application.

This kit is provided as individual enzymes to meet the customer’s needs and to provide maximum efficiency and flexibility in the RNA sample preparation. (Noted that these enzymes may still contain E. coli gDNA) The double stranded cDNA end product can subsequently be converted to blunt ended DNA fragments using abm’s DNA End Repair Kit (Cat. No. G477), followed by abm’s dA Tailing Kit (Cat. No. E009) to generate DNA suitable for whole genome sequencing.
Literature Click To Open
Application
  • RNA-Seq Library Construction.
  • downstream double-stranded blunt-end cDNA synthesis for cloning.
  • downstream double-stranded cDNA library construction.
  • Protocol Click To Open
    Storage Store all components at -20°C. All components are stable for 1 year from the date of shipping when stored and handled properly. Avoid repeated freeze-thaw cycles to retain maximum performance.
    Notes
  • one enzyme unit is defined as the amount of enzyme required to incorporate 1 nmol of deoxynucleotide into acid-precipitable material in 10 minutes at 37oC using Poly(A) and Oligo(dT) as template and primer, respectively.
    Noted that these enzymes may still contain E. coli gDNA.
  • Reaction Buffer Components: 20 mM Tris-HCl (pH 7.5), 12 mM (NH4)2SO4, 10 mM MgCl2, 0.16 mM β-NAD.
  • Product Documents
    Product References
    FAQs
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    Is OneScript® RT capable of strand switching?
    Due to mutations to remove RNase H activity in our OneScript® RT enzyme, the possibility of strand switching is very minimal.

    Non-mutated RTases with retained RNase H activity (such as AMV Reverse Transcriptase) are much more likely to ''strand switch''.
    What is the purpose of the dUTP kit over using dNTP?
    Generally the dNTP kit version is sufficient for most needs. You may wish to use the dUTP/dNTP kit sequencing information is required in a strand specific way.

    For example, if you use the dUTP, and dUTPase to digest the dUTP in the next step, all the sequence from the original mRNA will be removed. The sequencing information obtained in this case will ONLY be from the sense strand, NOT from the anti sense strand. This is sometimes required for special sequencing needs.