Hi,
two days ago I prepared competent E. coli JM109 by a protocol using 0.1 M CaCl2 solution. I grew the culture to an OD600 of ~ 0.5, spun them down and resuspended in chilled 0.1 M CaCl2. After a next spin I resuspended in CaCl2 and incubated for 1 h on ice to finally spin them down and resuspend in CaCl2 with 15 % glycerol. I aliquoted 100 µL in prechilled tubes which took a quite while (~ 100 tubes to be filled) and then I directly froze the tubes at -80°C. No freezing in LN2. After testing the Transformation Efficiency by pUC19 DNA I get only 5 colonies on the plate which is quite low. What possibly could have gone wrong? Happy about any suggestions!
Hi there,
Freezing method is an issue: the faster the better so dry ice or liquid N2 is absolutely required to preserve the cells. Did you actually run the assay right before and after freezing to compare and estimate the effect of freezig/thawing effect?
An other issue is the amount of DNA engaged. If too high the transformation efficiency is actually diminished. Ideally you should engage 0.1ng of plasmid (check the plasmid content of your plasmid stock right before the experiment). The efficiency is then extrapolated for 1 µg of plasmid. But if you actually run the experiment with 1µg the result will be far less...
Hi Dominique,
no I stored the cells at -80°C overnight and performed the transformation the next day with 0.01 ng pUC19 (stock = 10pg/µL). Besides the fact that the cells may be hampered by incubation in glycerol on ice the concentration of the Plasmid DNA may also matter. However, my group leader mentioned that this is too few colonies when 1 µL Plasmid DNA was used. Lets assume I put the cells in liquid Nitrogen after I added CaCl2 plus glycerol, how fast I have to put them to -80°C. And by the way, is 15 % glycerol enough for making competent E.coli?
Hi again,
Well your transformation efficiency is not that bad actually considering the lower limit is said to be 106/µg for cloning purpose. It might be that your are below the threshold just because of the freezing strategy (apparently you haven't flashfrozen your samples prior putting them in the -80°C). To answer your question, once the cells are in liquid N2 there is no timing to respect as the temperature is actually much lower than -80°C. 15% glycerol is enough and it is not actually used for making competent cells but to protect cells during th freezing process. The crucial point is to homogenize well cell suspension and glycerol prior freezing. And as I already said comparing efficiencies before and after freezing is a good point to evaluate the freezing process.
Hi,
this really helps. I scheduled the protocol for this week again and will consider your suggestions. Thanks so much!
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