We performed transformation on Saccharomyces cerevisiae, where we introduced a gene mutant library (roughly 14 mil. mutants). Because of the high transformation efficiency, many colonies appeared and created a lawn (the attached picture displays 1 out of 20 transformation plates).
It is very important to be able to calculate the exact transformation efficiency in order to estimate the size of the library.
Does anyone know a way to count the cells despite the 'lawn' formation?
Also, does anyone have any suggestions on how to stock the library?
(could it be as simple as scraping all the cells of the plate and making a glycerol stock?)
Thanks a lot for your help!
The first question you ask is how to calculate the numbers, in order to do so just plate some dilutions from the transformation stock (such as 10, 100, and 100-fold dilution) and count the colonies and extrapolate.
The second question regarding how to store, yes you can wash the plates with buffer and scrape up all the cells, then add glycerol and freeze all or it, or some portion of it (you don't really need everything).
Third I would point out that your plate looks like it was too wet when you plated and you have water streaks on the plate, this is not a major problem but might partially skew the population of cells that you stock away.
How do you get such high transformation efficiency of Saccharomyces cerevisiae? Can you share your experience?
Happy to answer your question @Dongwei Chen. Here is an excerpt of the methods section from this project:
'All S. cerevisiae transformations were performed according to the Lithium-acetate protocol (Gietz & Woods, 2002). Genes required for the use of the CRISPR toolbox were located on a plasmid and transformed in CEN.PK113-5D prior to any other transformation performed. The retention of the plasmid was ensured by growing the transformed strain on geneticin. A standard transformation consisted of an integration vector, acting as a repair template, and a gRNA cassette expressed on an episomal vector. The gRNA forms a complex with the Cas enzyme which creates a DSB in the genome, after which the integration vector is used as a repair template by HR mechanisms in the cell. The transformed cells were then inoculated on a selective solid media.'
Hope this helps!
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