silver sequence dna sequencing system  (Promega)

Journal: Methods (San Diego, Calif.)

Article Title: Analysis of post-transcriptional RNA metabolism in prokaryotes

doi: 10.1016/j.ymeth.2018.11.006

Figure Lengend Snippet: Analysis of 5' ends of leuX tRNA primary transcripts by primer extension [24]. All E. coli strains used in this study were derived from MG1693 (rph-1), which was considered wild-type. MG1693 has a single base pair deletion in the rph gene resulting in a frame-shift mutation [31]. The genotypes of the strains are noted at the top of the lane. Total RNA isolated from all the strains were reverse transcribed using a 32P-end labelled leuX tRNA specific primer and the cDNAs were separated on a 6% polyacrylamide sequencing gel. A leuX DNA sequencing ladder (CTAG) generated by same leuX tRNA specific primer was also separated along with the cDNAs and was used to identify the transcription initiation site (I) and the mature 5' end (II). The primer extension analysis also identified several RNase E cleavage sites (indicated as *) in the rnpA49 mutant, which disappeared in the rne-1 rnpA49 strain.

Article Snippet: The sequencing ladder is generated from the PCR DNA template by employing the same end-labelled primer used for reverse transcription ( section 6.1.1.2 ) using the Silver sequence™ DNA sequencing kit (Promega) according to the manufacturer instructions.

Techniques: Derivative Assay, Mutagenesis, Isolation, Sequencing, DNA Sequencing, Generated

Journal: Methods (San Diego, Calif.)

Article Title: Analysis of post-transcriptional RNA metabolism in prokaryotes

doi: 10.1016/j.ymeth.2018.11.006

Figure Lengend Snippet: Analysis of 5' ends of leuX tRNA primary transcripts by primer extension [24]. All E. coli strains used in this study were derived from MG1693 (rph-1), which was considered wild-type. MG1693 has a single base pair deletion in the rph gene resulting in a frame-shift mutation [31]. The genotypes of the strains are noted at the top of the lane. Total RNA isolated from all the strains were reverse transcribed using a 32P-end labelled leuX tRNA specific primer and the cDNAs were separated on a 6% polyacrylamide sequencing gel. A leuX DNA sequencing ladder (CTAG) generated by same leuX tRNA specific primer was also separated along with the cDNAs and was used to identify the transcription initiation site (I) and the mature 5' end (II). The primer extension analysis also identified several RNase E cleavage sites (indicated as *) in the rnpA49 mutant, which disappeared in the rne-1 rnpA49 strain.

Article Snippet: The sequencing ladder is generated from the PCR DNA template by employing the same end-labelled primer used for reverse transcription ( section 6.1.1.2 ) using the Silver sequenceTM DNA sequencing kit (Promega) according to the manufacturer instructions.

Techniques: Derivative Assay, Mutagenesis, Isolation, Reverse Transcription, Sequencing, DNA Sequencing, Generated

Journal: Methods (San Diego, Calif.)

Article Title: Analysis of post-transcriptional RNA metabolism in prokaryotes

doi: 10.1016/j.ymeth.2018.11.006

Figure Lengend Snippet: Analysis of 5' ends of leuX tRNA primary transcripts by primer extension [24]. All E. coli strains used in this study were derived from MG1693 (rph-1), which was considered wild-type. MG1693 has a single base pair deletion in the rph gene resulting in a frame-shift mutation [31]. The genotypes of the strains are noted at the top of the lane. Total RNA isolated from all the strains were reverse transcribed using a 32P-end labelled leuX tRNA specific primer and the cDNAs were separated on a 6% polyacrylamide sequencing gel. A leuX DNA sequencing ladder (CTAG) generated by same leuX tRNA specific primer was also separated along with the cDNAs and was used to identify the transcription initiation site (I) and the mature 5' end (II). The primer extension analysis also identified several RNase E cleavage sites (indicated as *) in the rnpA49 mutant, which disappeared in the rne-1 rnpA49 strain.

Article Snippet: The blue cDNA pellet is resuspended in 2.5 μl of Stop Solution from the Silver sequence™ DNA sequencing kit (Promega).

Techniques: Derivative Assay, Mutagenesis, Isolation, Sequencing, DNA Sequencing, Generated