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. 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.
RNA-Seq Library Construction.
downstream double-stranded blunt-end cDNA synthesis for cloning.
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.
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.
Reaction Buffer Components: 20 mM Tris-HCl (pH 7.5), 12 mM (NH4)2SO4, 10 mM MgCl2, 0.16 mM β-NAD.