It is important to practise sterile technique when growing bacterial cultures. In our project, we grew E. coli. cells.
All plasmids were miniprepped using the manufacturer’s protocol (the following protocol contains lab-specific details and notes):
This is a generic protocol for PCR reactions. It has been adapted in some of our other protocols for specific purposes, such as colony PCR and error-prone PCR.
All cloning work was carried out via restriction digestion and ligation. Typically, 250 ng – 1 µg of plasmid DNA and 250 – 500 ng of insert DNA was digested.
We produced chemically competent cells using rubidium chloride and calcium chloride.
E. coli. rendered chemically competent through treatment with calcium or rubidium chloride can be transformed with circularised DNA from isolated plasmids or from ligation products.
Purification of PCR products, restriction endonuclease digests etc. were carried out using the “PCR Clean-Up” protocol, and purification of DNA from agarose gels were carried out using the “DNA Extraction From Agarose Gels” protocol, both provided with the kit.
The protocols below have been adapted from the manufacturer’s protocol, with lab-specific details and notes.
Recombinant clones were screened using colony PCR, before positive colonies were picked for diagnostic digest.
Error-prone PCR generates random mutations in the amplified product of a target sequence. We did this by addition of manganese chloride to the reaction mix to cause Taq polymerase to lose its fidelity.
1. Wilson, D.S. and A.D. Keefe, Random Mutagenesis by PCR, in Current Protocols in Molecular Biology. 2001, John Wiley & Sons, Inc.
This method was used to analyse the different mutants of amilCP that were developed during this project.
Genes placed under the control of the T7 promoter can be inducibly expressed through the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG). We used this for expression of genes in recombinant E. coli. of the strain BL21 (DE3) with helper plasmid pLysS or pGro7.
Proteins expressed with a consecutive series of 6 histidines, or His6-Tagged, at the N or C terminus can be purified based on the affinity of histidine residues for divalent metal cations such as Ni2+ and Co2+. By passing cellular lysates through beads coated with these ions, the proteins of interest can be captured whilst the remaining cellular proteins are washed off. The tagged proteins can then eluted by the addition of a solution with a high concentration of imidazole (~300 mM) which competitively binds the nickel or cobalt, forcing the protein off.
Dialysis can be used to remove excess salts, such as imidazole, and also help to concentrate the protein sample.
Cellular proteins can be isolated through physical and chemical lysis or by other means of disrupting cell membranes. These proteins can then be separated electrophoretically through denaturing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) as proteins with different mobility, dependent on length and charge, will separate in size-dependent manner.
This assay tests for the binding of a certain protein to a certain segment of DNA through assessing the migration of the DNA through an electrophoresis gel. Protein-bound DNA should demonstrate band retardation. For our experiments, we used EtnR1 protein purified by his-tag using Dynabeads® followed by overnight dialysis.