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Protocol – Feeding Suspension Culture (Complete Replacement of Medium)
Complete replacement of culture medium from suspension culture involves cell harvesting by centrifugation and resuspension of the cell pellet in fresh growth medium. This method is ideally the best way to feed suspension culture and is often used when a. you observe dead or sick cells/suspended particles in culture; b. medium color (phenol red-containing medium) has turned yellow very quickly; c. you can not afford cell loss. Complete replacement of culture medium is ideally the best way to feed suspension culture. Since this procedure takes time and involves an additional centrifugation step to harvest cells, often researchers partially replace the medium that takes just a few minutes. However, partial medium replacement is associated with the loss of cells from the culture.
Protocol – Feeding Suspension Culture (Partial Replacement of Medium)
Replacement of culture medium from suspension culture is a bit tricky job as cells float in the medium. Ideally, cells are harvested from the medium by centrifugation and then resuspended in a fresh culture medium. However, in practice, a part of the culture medium is replaced with a fresh culture medium without harvesting the cells. This procedure is associated with the loss of cells.
Preparation of 10 M Sodium Hydroxide (NaOH) Solution
Here we have described a procedure to prepare 10 M (or 10 N) sodium hydroxide (NaOH) solution. A 10 M NaOH solution can be prepared by dissolving 40 g of NaOH (Molecular Weight: 40.00) in water to a final volume of 100 ml. Since dissolution of NaOH in water is a highly exothermic reaction, extreme care must be taken while preparing the solution.
Protocol – Thawing and Revival of Cryopreserved Cells
This protocol describes the revival process of cryopreserved adherent cells. The revival process involves rapid thawing of cryopreserved cells followed by removal of freezing medium which contains cryoprotectant, in this case, DMSO. The freezing medium can be removed immediately just after thawing the cells by centrifugation. Alternatively, cells are allowed to adhere to the culture dish followed by washing and addition of fresh growth medium.
Protocol – Preparing Liquid Culture of E. coli for Plasmid Miniprep
Overview:
Small-scale plasmid isolation, called plasmid miniprep, requires a small amount of bacterial culture. Normally, a 2 – 5 ml culture provides sufficient cells that can be processed for plasmid isolation using the miniprep protocol. Culture can be prepared by inoculating 3 ml antibiotic-containing growth medium with a single colony and growing it overnight at 37C with shaking.
Here we have taken an example of preparing liquid culture from a colony of E. coli DH5α, transformed with the pEGFP plasmid. The pEGFP plasmid contains a kanamycin resistance gene, therefore, requires kanamycin for the selection of plasmid-containing bacteria. If your plasmid carries another antibiotic-resistant gene, add the respective antibiotic to the culture medium.