From each sample of RNA (defined above, E. coli or murine total RNA, extracted and SHAPEmodified or untreated), 1-3 μg were subjected to MaP reverse transcription [requiring Superscript II and addition of Mn2+ to the RT buffer(25 (link), 39 (link))] using random nonamer primers. The cDNA generated was buffer exchanged (Illustra microspin G-50 columns, GE Healthcare) and the volume increased to 68 μL. For second-strand cDNA synthesis, 8 μL of 10× buffer (Second Strand Synthesis Reaction Buffer, NEB) and 4 μL enzyme (Second Strand Synthesis Enzyme mix, NEB) were added to the cDNA product and incubated for 2.5 h at 16 °C. Double-stranded cDNA was fragmented and amplified with sequencing indexes (Nextera XT library prep kit, Illumina). Nextera PCR products were affinity purified (using a 0.8× ratio of Agencourt AMPure XP beads, Beckman Coulter) and eluted in 20 μL of nuclease-free water.
Second strand synthesis reaction buffer
Manufactured by New England Biolabs
Sourced in United Kingdom
The Second Strand Synthesis Reaction Buffer is a buffer solution designed to facilitate the second-strand synthesis reaction in DNA sequencing and other molecular biology applications. It provides the appropriate ionic conditions and pH to enable the efficient synthesis of the complementary DNA strand. The buffer composition supports the enzymatic activity required for this step in the DNA synthesis process.
Automatically generated - may contain errors
Lab products found in correlation
Agencourt ampure xp bead, by Beckman Coulter (1 mentions)
Nextera xt library prep kit, by Illumina (1 mentions)
Illustra microspin g 50 column, by GE Healthcare (1 mentions)
Second strand synthesis enzyme mix, by New England Biolabs (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Promega (1 mentions)
Easypure plant rna kit, by Transgene (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
E coli dna polymerase 1, by New England Biolabs (1 mentions)
E coli ligase, by New England Biolabs (1 mentions)
E coli rnase h, by New England Biolabs (1 mentions)
2 protocols using second strand synthesis reaction buffer
1
Sequencing-Based RNA Structure Analysis
2
Total RNA Extraction and cDNA Synthesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Total RNA was extracted from ca. 100 mg of floral buds or leaves, with Trizol Reagent (Thermo Fisher Scientific, Waltham, Massachusetts, USA) following manufacturer's instructions, and re-purified using either RNeasy Plant Mini Kit (QIAGEN) or EasyPure Plant RNA kit (Transgen Biotech, Beijing, China) . After verification of RNA integrity by agarose gel electrophoresis (1 % w/v), RNA was treated with 4 U of DNase I (Promega, Madison, Wisconsin, USA). Simple-stranded cDNA was synthesized using the SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific) and oligo dT 20 as the primer, following the manufacturer's protocol. Then, double-stranded cDNA was obtained in a 150 µl-reaction containing 1X Second Strand Synthesis Reaction Buffer (New England Biolabs, NEB, Hitchin, Hertfordshire, UK), 2 mM dNTP mix, 10 U of E. coli ligase (NEB), 40 U of E. coli DNA polymerase I (NEB), 2 U of E. coli RNase H (NEB) and DEPC-treated water. The mix was incubated for 2 h at 16 ºC. Double-stranded cDNA was purified with one volume of phenol:chloroform:isoamyl alcohol (25:24:1), precipitated with NH 4 OAc 7.5 M and resuspended in 10 mM Tris-HCl.
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