Taq dna ligase buffer

Single-stranded oligonucleotides (in Tables 1 and S1†) were purchased from GENEWIZ (Suzhou, China). The sequence of l-DNA₅₉ was a part of l-DNA₇₄, which was from phage M13, and their secondary structures were determined by Mfold calculation.^{33 (link)} Prior to the cyclization experiments, a phosphate residue was enzymatically introduced to the 5′-position of these linear DNAs (l-DNAs) by using T4 polynucleotide kinase (Thermo Scientific, Pittsburgh, PA. USA). The products were purified by PAGE. Thermus aquaticus (Taq) DNA ligase was obtained from NEW ENGLAND BioLabs (Beijing, China), together with 10× Taq DNA ligase buffer. T4 DNA ligase was purchased from Thermo Scientific (Pittsburgh, PA. USA), together with 10× T4 DNA ligase buffer. Exonuclease I was from Thermo Scientific (Pittsburgh, PA. USA). All other chemicals were from Sigma-Aldrich (St. Louis, MO. USA).

The fluorogenic glycine riboswitches were constructed from four oligonucleotides O1–O4 (Sigma Aldrich, Germany). O1 contained a T7 promoter and a DNA-pull down anchor sequence. O2 and O3 encoded the natural glycine sensor aptamer 1 and 2 from B. subtilis, respectively. O4 carried the variable transmitter and a previous functionally optimized Spinach actuator sequence. All oligonucleotides in Supplementary Table S1 were ordered from Sigma Aldrich (Taufkirchen, Germany). For the linkage of the complete riboswitch sequences, O1-O4 were combined by POS. In a POS reaction (see Supplementary Figure S1), O1–O4 were mixed with connectors C1–C3 at an equal concentration of 1 μM in Taq DNA Ligase Buffer (New England Biolabs, USA). Only O4 and C3 were specific for each riboswitch. Upon addition of 40 U Taq DNA Ligase per 25 μl reaction vessel, the reverse strand of the fluorogenic glycine riboswitch were ligated together. Constructs of the negative control for all 94 riboswitches were produced in a single POS reaction by omitting the fragments O2 and O3. Purified polymerase chain reaction samples were dissolved in 1x SSC buffer and 1% w/v BSA at a DNA concentration of ∼90 ng/ml and spotted onto an epoxy coated microscope slide (Cel-1, USA) with a microarray contact printer (OmniGrid).