User:Barry Canton/Notebook/T7 RNAP transcription of rRNA

Introduction

I have been transcribing rRNA with T7 RNA polymerase in E. coli. I would like to estimate how much immature rRNA transcript I am producing relative to that being made on the chromosome. I would also like to estimate how efficiently the T7-transcribed rRNA is being incorporated into active, functional ribosomes.

Milestones

1. Specific detection of wild-type and mutant 16s rRNA
   • Design 3 sets of primers (use Bryan's design first). Order Monday
   • Do extraction on Wednesday?
   • Do blot on Wednesday/Thursday.
   • Pick best primer pairs
2. Relative quantification of wild-type and mutant 16s rRNA
3. Evaluation of efficiency of processing of mutant rRNA relative to wild-type rRNA.
4. Write & Submit paper.

Protocols

• Sauer:RNA Purification from E. coli
• Sean's RNA Purification protocol - short
• Northern Blotting
• Polysome gradient running
• List of Supplies

Related protocols

• Native and denaturing lysis protocols collected from OWW.
• P1 transduction to make the rna knockout

Recent changes