I had prepared electro competent cells (in 10% glycerol) of Mycobacterium smegmatis to inactivate a gene using electroporation a mode of transformation and homologous recombination a method for allelic exchange. I have noticed, freshly prepared competent cells work fantastically for transformation but the stored (in -80C) one's are not. I was just wondering, do they die due to cold shock or there may be other reasons?
Hi there,
Freezing of electrocompetent cells for M.smegmatis is well-documented. Then there must be something wrong with your freezing/storage/thawing conditions. Do you flash freeze cells in liquid nitrogen or dry ice? Do you thaw aliquots on ice? How dense is the cell suspension prior to freezing? As only a very small fraction of cells will survive to freezing/thawing cycle and if not dense enough your prep won't give you enough survivors to finally get transformants.
Cells are visually dense. I do not flash-freeze cells in liquid nitrogen or dry ice rather i thaw them in ice for about 30 min.
Rapid freezing process is essential to maintain viability at a level suitable for downstream efficient transformation. So if you want to generate frozen stock of competent cells, use liquid nitrogen or dry ice for freezing samples.
- Badges
- Science topic
- Similar topics
- Geoscience
- Cartography
- Mapping