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Freezing home-brewed competent cells


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6 replies to this topic

#1 seanspotatobusiness

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Posted 24 January 2011 - 01:55 PM

I have a protocol that states that newly-made competent cells should be first frozen in dry ice and then transferred to storage at -80 C. Why is this? Will it produce a quicker or slower cooling? Another source suggested a dry ice/ethanol bath.

I don't have a reliable source of dry ice, so I'd rather find a way to do it without. If they need to cool quickly, how about a metal heat-block, sitting in the -80 C? Else, slowly, how about a polystyrene enzyme box thing? You know the things I mean? They keep your microcentrifuge tubes all snug in those white plastic boxes.

#2 bob1

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Posted 24 January 2011 - 02:36 PM

The dry ice is supposed to cool more rapidly... however by itself, it is just as slow as the -80 (and slightly warmer too, it slowly sublimes if kept in the -80). Usually if you want faster cooling you need to make a dry ice/ethanol or methanol or isopropanol bath. I have never fully tested it, but have never had a problem just putting freshly made competent cells straight into the -80.

#3 vetticus3

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Posted 25 January 2011 - 02:37 AM

the sooner they are frozen, the better quality they are. tested in my hands from the same batch, one vial frozen in dry ice straight away, the other kept on ice until i could walk to the -80'c freezer. both were good quality, but the dry ice were better. i liken it to frozen vegetables - you have to lock in the goodness.

I think it also has to do with how far you have to walk to the -80'C.
in my old lab, it was in the basement, and we were on the 5th floor - the lifts didn't always work and the stairs were very steep. so, if the -80'c was next to you - no problem.

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#4 HomeBrew

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Posted 25 January 2011 - 03:53 AM

I used to religiously use a dry ice-ethanol bath to freeze competent cells before transfer to the -80, but a few years ago I skipped it, saw no difference in the competence of those cells, and so now I don't bother any more. Haven't had a transformation problem since I switched.

#5 seanspotatobusiness

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Posted 26 January 2011 - 04:28 AM

Hi guys, thanks for the advice. I've got readier access to liquid nitrogen than to dry ice (which requires a little more forward planning). Do you know whether that would be just as good as the ethanol-dry ice bath?

#6 HomeBrew

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Posted 26 January 2011 - 04:53 AM

I don't know about using liquid nitrogen -- I never tried it (or know of anyone who has). Why not just keep them on ice and put them straight in the -80, as we've all suggested?

Think of it this way -- if a short period on ice were going to effect competency much, then most of our transformations would fail because the cells invariably sit on ice for a while when you pull them out of the -80 to use them.

#7 seanspotatobusiness

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Posted 26 January 2011 - 05:35 AM

Well, Veticus suggested that the quality of his cells were improved by freezing on dry ice, and an NEB representative whom I contacted (about something else) also underlined the supposed importance of fast freezing with dry ice. Thus, based on all your input, I conclude that acceptable efficiency is acheived by just putting them at -80 C while superior efficiency is acheived by fast-freezing. Since I'm going to try to write the SOP for our group, it would be useful to include this information for people who have need for super-high efficiency cells, even if I don't actually need to do that myself. I may well do both and see for myself the difference with a pUC transformation, in which case I'll report back here.




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