SOPs

This method describes how to derivatize the N-glutathionylspermidine and trypanothione produced by T. brucei trypanothione synthetase under in vivo-like conditions

This is a protocol for determining the activity of the T. brucei Trypanothione synthetase under in vivo-like conditions.

This method describes the preparation of the in vivo-like buffer for the measurement of bloodstream T. brucei recombinant enzymes under pseudo-physiological conditions.

Sample preparation procedure for metabolic analysis on T. b. brucei 427 using LC/MS

Labelling and extraction procedure for uniformly 13C-labelled E. coli (MG1655) for absolute quantification using isotope dilution technique by LC-MS

Sample preparation procedure for metabolic footprint analysis on T. b. brucei 427 using LC/MS

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

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′ ...