Protocol for transfer of plasmids into Clostridium
acetobutylicum ATCC 824 by electroporation
Contributor: Ying Zhang
The phosphorylation level of a particular protein can be determined using a procedure based upon western immunoblotting, with Phos-tag™ reagent present in the SDS-PAGE gel. The Phos-tag™ reagent, supplied in the form of Phos-tag™ acrylamide (Wako Pure Chemical Industries, AAL-107), causes proteins to be resolved both on the basis of size and phosphorylation state. This means that phosphorylated and de-phosphorylated forms of the same protein can be distinguished.
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 used for detecting non influential parameters and interactions in non linear dynamical models. These parameters can be estabilished which allows the prioritization of parameters that can be subsequently estimated using robust global optimizations methods.
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.
A procedure to measure the levels of GSH and SLG before and after exposure to MG using formic acid. A similar expreimental set up as for potassium efflux experiments is used to which a silicon oil centrifugation step is incorporated. Samples are analysed by LC-MS-MS in order to quantify intracellular GSH and SLG levels.
Cells were harvested from culture keeping the cells cold to quench the physiological condition of RNA and the cells were mechanically disrupted. RNA was isolated from the cells by conventional acid-phenol method and the quality was checked by Agilent bioanalyser.
The reverse transcriptase synthesizes DNA, which complements the mRNA template
(complementary DNA, cDNA). Cy3/Cy5-dCTP are incorporated into cDNA during Reverse transcription. The obtained Cy3/Cy5 cDNA are then competitively hybridised onto Agilent microarray slide and subsequently scanned.
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.
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
Isolation of total RNA from Bacillus Subtilis using phenol-chloroform extraction method by maintaining cryogenec conditions initailly to prevent RNA degradation. Quality of the obtained RNA is then tested with Agilent Bioanalyser before proceeding for gene expression analysis.