Adenine binding aptamer green (MG) binding aptamer cassette 3’ Table 1: T-box-linker-aptamer sequence. The yellow highlights are the restriction sites. Cloning cassettes into pGEM-3Zf(+) for transcription Restriction analysis and gel purification: The custom plasmids from IDT were digested using EcoRI and PstI and cleaned using Qiagen PCR purification kit. The digested product was run on a 2% low melt agarose gel and bands corresponding to each released insert were excised and the DNA purified (Qiagen gel extraction kit). Purified DNA was checked on 1% agarose gel (Figure 7; result section). Ligation and Transformation: The DNA fragments, T-box-MG aptamer, T-box-2AP aptamer, 2AP aptamer, were ligated to and PstI cut pGEM-3ZF plasmid using 200 U (in a 10 reaction) of T4 DNA μL ligase. Using the heat shock protocol, 2 µL of each ligation mix was used to transform 50 µL of XL-10 competent E. coli cells. The cells were plated on LB+ampicillin (100μg/ml) plates and grown overnight at 37 °C. DNA purification and restriction analysis to look for presence of proper insert: Picked 4 colonies from each plate and inoculated 3 mL LB+ampicillin. Grew the cells overnight at 250 rpm at 37 °C. Purified the plasmid DNA using Qiagen spin miniprep kit
Adenine binding aptamer green (MG) binding aptamer cassette 3’ Table 1: T-box-linker-aptamer sequence. The yellow highlights are the restriction sites. Cloning cassettes into pGEM-3Zf(+) for transcription Restriction analysis and gel purification: The custom plasmids from IDT were digested using EcoRI and PstI and cleaned using Qiagen PCR purification kit. The digested product was run on a 2% low melt agarose gel and bands corresponding to each released insert were excised and the DNA purified (Qiagen gel extraction kit). Purified DNA was checked on 1% agarose gel (Figure 7; result section). Ligation and Transformation: The DNA fragments, T-box-MG aptamer, T-box-2AP aptamer, 2AP aptamer, were ligated to and PstI cut pGEM-3ZF plasmid using 200 U (in a 10 μL reaction) of T4 DNA ligase. Using the heat shock protocol, 2 µL of each ligation mix was used to transform 50 µL of XL-10 competent E. coli cells. The cells were plated on LB+ampicillin (100μg/ml) plates and grown overnight at 37 °C. DNA purification and restriction analysis to look for presence of proper insert: Picked 4 colonies from each plate and inoculated 3 mL LB+ampicillin. Grew the cells overnight at 250 rpm at 37 °C. Purified the plasmid DNA using Qiagen spin miniprep kit
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