I’m trying to reproduce the library transformation efficiencies seen in this 2010 paper: Article An improved yeast transformation method for the generation o...
in which they claim 1.5 x 10^8 cf/ug DNA. My intact circular vector is the same size as their pcr product + linearized vector, but I’m getting 10^5 ish transformants/ug, similar to chemical transformation with the zymo kit.
Has anyone successfully done this? so far we’ve tried different electroporators, voltages, recovery media, etc but nothing seems to be working.
Any other ideas or troubleshooting would be much appreciated, thanks in advance
Look, unless your goal is to make the transformation more efficient, just plate out more cells from the transformation.
Hi Timothy K. Craig
There could be several reasons why you're not achieving the same transformation efficiency as the paper. Here are a few things to consider: Quality of Plasmid DNA: Ensure that your plasmid DNA is of high quality, pure and not degraded. Use a reliable method to isolate and purify your DNA. Competency of Yeast Cells: The competency of yeast cells is critical. Make sure that the cells are appropriately prepared, not too old or too young, and handled gently during the preparation process. Plasmid DNA Concentration: The amount of DNA used can impact transformation efficiency. Verify the concentration of your plasmid DNA using a reliable method such as spectrophotometry or Qubit fluorometer. Transformation Protocol: Be sure you're following the protocol closely. Small details, like the temperature and timing of heat shocks or the voltage and cuvette gap in electroporation, can make a big difference. Media and Growth Conditions: The choice of media, pH, temperature, and even the type of agar can affect transformation efficiency. Strain Variation: Different yeast strains can have different transformation efficiencies, even with the same plasmid. Make sure your yeast strain is identical to that used in the paper. Experiment Reproducibility: Even when a protocol is followed precisely, achieving the exact same results can be challenging due to variability in conditions and materials.
I would check the quality of my plasmid and also check that your competent cells are all viable. it would be useful to plate them on a medium without selection to see if they all grow or not.
I don't think it's a protocol issue, but I've worked with this one before and it's a good one:
https://www.nature.com/articles/nprot.2007.15
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