Goscript rt pcr kit

The transcription start site for genes lvaR and lvaA were isolated using an adapted 5’ Race protocol from Schramm et al(Schramm et al., 2000 (link)). The RNA isolated from P. putida KT2440 was treated with the TURBO DNA-free™ Kit from Ambion^® to remove any contaminating DNA. The Promega GoScript RT PCR kit was used to generate cDNA using 1 µL of a 10 µM gene specific oligo (JMR2 for lvaR and JMR287 for lvaA) instead of the random oligo mixture. Following the inactivation of the reverse transcriptase, the cDNA was purified using Qiagen PCR Purification kit. Tailing of the cDNA was achieved using the terminal deoxynucleotidyl transferase (TdT) enzyme from Thermo Scientific. The final reaction mixture contained 1× reaction buffer, 1 pmol cDNA fragments, 60 pmol dGTP or dCTP and 30 U TdT. The reaction was incubated at 37°C for 15 min and then quenched by heating to 70°C for 10 min and the tailed cDNA fragments cleaned up using a Qiagen PCR Purification kit. The tailed cDNA was amplified using GoTaq Green Master Mix with an annealing temperature of 55°C and an extension time of 30 sec. Primer GG318 was used for dGTP tailing and ALM244 was used for dCTP tailing. The reverse primer for lvaR was JMR150 and for lvaA was JMR296. The resulting PCR product was submitted for sequencing.

Using the DNAse treated RNA isolated from LA grown P. putida KT2440, cDNA for the operon was generated with the Promega GoScript RT PCR kit using 1 µL of a 10 µM gene specific oligo (JMR237). The cDNA was then used as the template for PCR reactions using GoTaq Green Master Mix with an annealing temperature of 55°C and an extension time of 0:30 sec. Primers used for each gene are given in Supplementary Table 8.