I would like to know if there is any protocol or personal experience to cryopreserve chemically competent E.coli cells (for routine subloning) at -20C. The only ultra-freezer that I have access is about an hour driving from my lab. Thanks!
The survival of Escherichia coli decreased after week 16 of freezing at -20oC, but organism survived under cryogenic preservation (-80oC) until week 18. See the reference below:
Onderstepoort j. vet. res. vol.79 n.1 Pretoria Jan. 2012
Comparing effects of freezing at -196 ºC and -20 ºC on the viability of mastitis pathogensInge-Marie PetzerI et al. ABSTRACTThe aim of this study was to compare the effects of cryopreservation at approximately -196 ºC in liquid nitrogen (N) and freezing at approximately -20 ºC in a freezer, on the viability and survival of eight different mastitogenic bacteria inoculated in milk. Bacteria were frozen at approximately -20 ºC in a freezer and cryopreserved at approximately -196 ºC in liquid nitrogen. An effective preservation method was needed for follow-up samples from cows identified in the South African National Milk Recording Scheme (NMRS) with somatic cell counts above 250 000 cells/mL milk. The organisation responsible for sample collection of the NMRS milk samples also provides producers with liquid nitrogen for their semen flasks at the collection sites. This existing mode of storage and transport could therefore be utilised. Ten samples of each organism were thawed and cultured bi-weekly until week 18 for both temperature treatments. An additional sampling was performed at week 30 for samples frozen at approximately -20 ºC. Freezing and cryopreservation did not impair subsequent isolation of Streptococcus dysgalactiae, Streptococcus uberis, Enterococcus faecalis, Staphylococcus aureus (STH) (phage type lytic group III) or Sta. aureus (STA) (phage typed, other than lytic group III). Survival was indicated by the isolation of bacteria from samples, and viability by the strength of growth of the bacteria isolated. The survival of Streptococcus agalactiae decreased after week 12 and Escherichia coli after week 16 of freezing, but both organisms survived under cryogenic preservation until week 18. Coagulase-negative staphylococci survived until week 18 for both freezing and cryogenic preservation. Both storage methods could thus contribute to the improvement of a pro-active approach towards udder health management in South African dairy herds.
Hello, see reference attached.
Only thing after cryoconservation for recovery of culture use fast hitting, just put tube direct to 37 *C water bath.
Hello,
For chemically competent cells I have not seen any protocols that utilize cells frozen at -20°C most likely due to decrease in efficiency or viability. However, there is a report that describes room temperature electrocomptent cells that might be of use if you have access to an electroporator (they can be stored as a dry pellet for 3 days at 4°C). find the method here: https://www.nature.com/articles/srep24648. if you must use chemically competent cells you can instead use this protocol to make them (this will not help you with the freezing issue, however, it is the quickest protocol to make competent cells for routine use and may lessen the pain of making the cells). Find the protocol here: https://openwetware.org/wiki/TSS. Finally, I would suggest to actually try either the cells you have or the ones made with the TSS method at -20°C. You can make the following modification when transforming cells that are sitting at -20°C. Mix 100ul of your cells + 10ul of DNA (or ligation rxn, but preferably test this with a plasmid first like pUC19) then continue your normal protocol. the large amounts of cells and plasmid might make up for the reduction of efficiency from freezing at -20°C.
Best of luck!
Hanna
- Badges
- Science method