We extended our HighYield T7 RNA Labeling Kits with an additional reaction buffer to increase optimization flexibility for individual assay set-up.
Optimal reaction conditions for maximizing product yield and labeling density strongly depend on the nucleotide analog and the type of template used. Adjusting the ratio of labeled to unlabeled NTPs is a key optimization parameter, and can be readily fine-tuned using our modular kit configuration.
The additional HighYield T7 LabelOpt Buffer offers another option to fine -tune assay conditions e.g. for intrinsically inefficient enzymatic substrates such as DNP-11-UTP (Fig. 1).
Figure 1: HighYield T7 LabelOpt Buffer increases product yield of inefficient enzymatic substrate DNP-11-UTP.
Visualization of RNA with intercalator EtBr. 0 – 100 %: Increasing percentage of UTP substitution by DNP-11-UTP.
1.4 kbp RNA was generated using HighYield T7 AZDye488 RNA Labeling Kit components in combination with DNP-11-UTP and HighYield T7 Reaction Buffer or HighYield T7 LabelOpt Buffer.
Reaction conditions: 100 ng DNA template, 2.5 mM each ATP, CTP, GTP, 0.5 mM total UTP (UTP & DNP-11-UTP), 30 min at 37°C. Crude in vitro reaction mix was subsequently analyzed by agarose gel electrophoresis. M-215: 200 bp DNA ladder
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