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| Restriction Digest with NEB enzymes |
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Restriction enzymes (REs) are ‘molecular scissors’ that can be used to cleave DNA at specific, palindromic sequences. In our lab, REs that create single-stranded overhangs are employed to create ‘sticky ends’. Hence, we can cut specific sequences of DNA and subsequently join them together.
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| PCR Purification |
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Polymerase chain reaction (PCR) allows for the amplication of double stranded DNA sequences. After PCR, DNA product can be run on a gel to check if the fragment is of the correct size, and subsequently purified for downstream applications.
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| Transfection-Expression |
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Bacteria can be transformed with plasmids of interest for larger scale production of plasmids, and expression of genes of interest. We use calcium competent cells, which are easily transformed using the heatshock method.
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| Bacteria Culture |
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Bacteria work is a major component of our lab. While we only work with Risk Group 1 agents, proper working practices are nonetheless needed to ensure lab safety and reliable experimental results.
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| Cell Number and Viability Determination |
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During maintenance of mammalian cells, it is recommended that cell viability and concentration be measured and recorded. A small volume of mammalian cells in culture media can be diluted with equal volume of trypan blue dye, which stains dead cells, and the cell concentration calculated with the use of a haemocytometer.
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| Freezing Down Cells |
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Mammalian cell cultures should be frozen down and banked at low passage numbers. At higher passage numbers, cell cultures may lose phenotypic behaviour and become unhealthy. Mammalian cells banked at earlier passage numbers can be revived regularly and old cultures discarded to ensure reproducibility.
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| Making Bacteria Media |
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Bacteria are cultured in our lab, with the use of Luria Broth (LB) for growth in suspension and LB agar for growth on solid media.
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| Miniprep of Plasmid DNA |
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Bacteria stocks carrying plasmids can be grown in small volumes and lysed, to extract plasmid DNA for cloning purposes.
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| Subculturing for Adherent Cell Lines |
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Mammalian cells must be subcultured and maintained according to their doubling times. Refer to ATCC for recommended media and doubling times. The two cell lines we use, HEK293 and HepG2 are maintained in DMEM supplemented with Fetal bovine serum.
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| Ligation Protocol with T4 DNA Ligase |
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T4 DNA ligase is able to join both blunt and sticky ends. This allows us to ligate insert DNA of interest into circular vectors for stable expression of genes of interest.
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Antibiotic Preparation |
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Antibiotics are commonly used as selection markers for bacteria which are carrying plasmids of interest. Preparing sterile antibiotics is essential for correct selection in cloning.
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| Mammalian Cell Invasion Assay |
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Bacteria can invade mammalian cells upon the expression of secreted proteins, that enable penetration of the cell membrane. The mammalian cell invasion assay allows for characterisation of this phenotype.
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| Agarose Electrophoresis |
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Agarose gel electrophoresis is the standard way of separating and analysing DNA of mixed fragment size. When a current is applied to an agarose gel, a potential difference is created at either ends of the gel. DNA, which is negatively charged will hence migrate in the gel, and be separated according to size, as larger fragments migrate at slower speeds.
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