Team:Exeter/Protocols
This page displays the list of protocols used in the wet lab. Many protocols, such as transformations or competent cell preparation, were used at multiple times throughout the project. This list does not include InterLab.
Protocol |
Description |
Link |
|---|---|---|
| Preparation of competent E. coli cells | DH5α and BL21(DE3) E. coli strains used throughout the project were made chemically competent by following this protocol. By preparing competent cells as a team rather than purchasing commercial cells this reduced costs as well as giving the team basic lab skills. | Comp Cells | Transformation of E. coli homemade competent cells | This protocol was used a lot over the summer, due to the multiple DNA constructs worked with. The uptake of plasmids in different E. coli strains was vital to proving the theory of the project worked. | Transforming |
| Modular Cloning Strategy (One Pot cloning) | Also known as 'one pot' or 'MoClo' cloning, this method results in the direct assembly of DNA parts using restriction enzyme BsaI. Up to six DNA fragments in a single backbone can be assembled this way which was useful when attempting to build large constructs for this project. | One Pot Cloning |
| DNA Plasmid Miniprep | This protocol extracts and isolates plasmid DNA from bacteria, allowing the team to extract the plasmids containing the chlorite dismutase and perchlorate reductase gene constructs and ensure that they are present in the transformed E. coli cells. | Miniprep |
| Qubit DNA assay | A Qubit DNA assay was performed after every DNA MiniPrep extraction to find the concentration of DNA in the samples. This is useful to show if there is enough DNA present to send for sequencing and if the MiniPrep was successful. The Qubit reader uses fluorescent dyes to produce a signal which identifies DNA concentration. | Qubit |
| DNA Fast Enzyme Digest | An enzyme digest to allow the team to extract the cld gene to run through an agarose gel, below. | ThermoFisher Fast Digest Restriction Enzyme kit |
| IPTG Induction of E. coli | This protocol is for the rapid growth of E. coli from overnight cultures. | iPTG Induction |
| Bug Buster Protein Extraction Reagent | This Novagen protocol lysed the E. coli cells containing the signal peptide GFP's and the Chlorite dismutases to release the proteins. It meant the team could analyse the proteins through the mini gel electrophoresis, below. | Merck Millipore BugBuster |
| Western blot transfer | The protocol allows the proteins from the Bolt Bis-Tris gel electrophoresis, above, to be transferred from the SDS-page gel to a PVDF membrane in preparation of the Western blot. | Western blot Transfer |
| iBindTM Western System | An iBind Western Device produces a faster, cleaner, automatic Western blot compared to traditional methods and was performed to demonstrate the presence of cloned proteins. | iBind Western |
| Fractionation of E. coli | This protocol separated the periplasm from the cytoplasm of the E. coli cells containing signal peptide GFP sequences, allowing the team to demonstrate that the signal peptides are crossing the membranes. | Fractionation |
| Bolt Bis-Tris Plus Mini Gel Electrophoresis protocol | This protocol runs the sample from the Bug Buster protein protocol down a gel to demonstrate the presence of differently sized proteins and show that cloning of the signal peptide GFP's and Chlorite dismutases into E. coli had been successful. | Gel Electrophoresis |
| Perchlorate assay | An assay developed by the team, this protocol was sent to teams around the world in the hopes of testing for perchlorate in areas with high concentrations. It has been revised multiple times, and requires strict health and safety requirements. | Perchlorate assay |
| Clark Electrode | The Clark Oxygen Electrode measured the oxygen production of E. coli cell lysate containing chlorite dismutase from several species in the presence of chlorite solution. | Clark electrode |
| Preparing LB plates | This protocol was used to prepare any plates needed including growing colonies after transformations and picking colonies for overnight cultures. | LB plates |
| Overnight cultures | Overnight cultures; this protocol is for the preparation of overnight cultures containing antibiotic to allow bacterial cell growth. | Overnight cultures |