SOPs

Assay methodologies for individual glycolytic isoenzymes from the Mendes Group, University of Manchester, UK

This is the general protocol for the glycolytic enzyme measurements. Detailed Information for each Enzyme can be found in the SOP: Assays for measuring the activities of the individual glycolytic isoenzymes of Saccharomyces cerevisiae

For the study of mRNA decay rates, transcription was inhibited with ActinomycinD, and RNA splicing with Sinefungin, at different time points, in the Matthews lab. rRNA depleted RNA was extracted from each of the samples in the Clayton lab, and sent for deep sequencing at the BioQuant facility in Heidelberg

Protocol for transfer of plasmids into Clostridium acetobutylicum ATCC 824 by electroporation

ClosTron mutants should always be subjected to Southern blot analysis to ensure that only one intron insertion has occurred.

A protocol to improve conventional, recombination-based gene knock-out methodologies thtough the provision of negative selection markers, pyrE or codA.

Protocol for transfer of plasmids into Clostridium spp. by conjugation

A refined and streamlined procedure to generate mutant in a wide range of different clostridial species, using group II intron retargeting methodologies.

This protocol describes the transcriptional profiling of E. coli cultures using microarrays. The protocol utilises RNA isolated as described in another SOP (SUMO RNA isolation from E. coli) and with hybridisation to Ocimum Ocichip E. coli K-12 microarrays.

This SOP describes the preparation of E. coli RNA

SOP for growing yeast in anaerobic conditions

Biomass sampling - SOP for sampling of biomass

Metabolic perturbations - SOP for metabolic perturbations (i.e. glucose pulse)

Yeast strains used in the project

General protocol for measuring the kinetic parameters of the purified glycolytic enzymes from Saccharomyces cerevisiae - SOP for measuring the kinetic parameters of the purified glycolytic isoenzymes

Sample preparation - SOP for sampling, preparation of cell-free extracts, and determination of total extracted protein

Sample preparation - SOP for sampling, preparation of cell extracts, and general assay set-up

We routinely select specific RNAi gene targets (400–600 bp) and primers using the RNAit software http://trypanofan.path.cam.ac.uk/software/RNAit.html. A single pair of PCR primers are designed that incorporate four selected restriction sites (not present in the RNAi target fragment) such that a single PCR product can be differentially digested and sequentially cloned. For example, using MCS1/2, the following primers could be used to clone antisense followed by sense fragments: Primer 1, XbaI–BamHI-5′ ...

SOP for extracellular metabolite measurment

Written Standard Operating Procedures provide workers with the operational information necessary to perform a job properly and ensure consistency in the operations. Standard Operating Procedures provide a historical record of steps in the how, why and when and serve as a training tool for teaching users.

The kit is used for the quantitative detection of ATP in yeast cells by luciferase driven bioluminescence. ATP extraction is done by boiling cells in TE buffer.