In the past decade, RNA sequencing has become a notable method for identifying transcript alterations that correlate to disease development and progression. RNA-seq allows researchers to pinpoint mutated sequence elements that hinder transcriptional regulation, ultimately altering cellular functioning, and specify research questions around disease advancement. To use RNA-seq as a reliable molecular tool for mapping RNAs in diseased cells, researchers have focused on optimizing the step in which an adaptor is ligated to the RNA to be sequenced, prior to cDNA development. Although wildtype T4 RNA Ligase 2 (Rnl2) has been previously utilized for this step, its usage develops a mixture of ligated products and circularized RNA, making it an unreliable tool. C-terminus truncated T4 RNA Ligase 2 (1-249) with double mutations K227Q and R55K (DM Rnl2trunc) has been identified as an efficient tool for ligating known pre-adenylated adaptors to any RNA with reduced amounts of unwanted side products. In this study, we present the experimental parameters utilized to successfully purify a high yield of DM Rnl2trunc that is both soluble and enzymatically active from E. coli cultures. Growth media volume was identified as the largest contributing factor for solubilizing DM Rnl2trunc. Further, SDS-PAGE analysis suggests the recombinant protein was eluted from nickel columns using 100mM imidazole. Most importantly, our purification protocol yielded abundant DM Rnl2trunc with enzymatic activity comparable to commercially available product. Bioanalyzer data indicates both homemade and commercial DM Rnl2trunc is capable of ligating desired RNA sequences and pre-adenylated adaptors with very high efficiency and unwanted side products.