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best freezing method for electrocompetent cells


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#1 azim58

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Posted 05 May 2011 - 02:53 PM

I am making electrocompetent cells, and I need to obtain the highest transformation efficiency possible since I am making a cDNA library. I am wondering what would be the best freezing method at the end of the protocol to make the cells competent (centrifuging a bunch of times to remove the salts in preparation for electroporation). Basically, there are 3 methods I could use:

1. Flash freeze (aka snap freeze) the cells by dropping the tube of cells into liquid nitrogen and then placing them into the -80 C.
2. Dry ice ethanol bath. Place the cells in the dry ice ethanol bath for a little while, and then transfer to the -80 C.
3. Cryofreeze container with isopropanol. Place the cells into a cryofreeze container with isopropanol and put this container into the -80 C freezer. With this method, the temperature would decrease very gradually (-1 C per minute).

In most protocols I look at, I see the flash freeze method listed. However, then I came across some directions in a commercial electrocompetent cell box (DH10B cells from Invitrogen). In these instructions, they said that for best results after one used the cells for transformation not to put them directly back into the -80 or to flash freeze, but to put the cells in a dry ice ethanol bath for 5 minutes and then place them into -80 C. These instructions made me think that flash freezing may be harder on the cells and that maybe I should not flash freeze when making the cells competent if I want the highest transformation efficiency.

Now I am leaning towards the cryofreeze method people use with mammalian cell culture. This seems to be the gentlest method.

#2 protolder

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Posted 05 May 2011 - 09:30 PM

Hola,Only one thing, I freeze aliquots in dry ice, but to avoid temperature changes I have the aliquots tubes cold in dry ice(I think that you have cold tubes too)Buena suerte y eficiencia

#3 azim58

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Posted 06 August 2011 - 05:11 PM

I just did an experiment which compared two of the freezing methods after preparing electrocompetent cells. I had a "slow freeze" group (tubes placed into an isopropanol cryogenic freezing container so that the temperature dropped 1 C per minute down to -80 C), and I also had a "fast freeze" group (tube dropped into liquid nitrogen and then placed into -80 C freezer). I also transformed with two different plasmids. I did not find a large difference between the two different freezing methods, but the fast freeze method did yield slightly higher transformation efficiencies. The results are below.

Freezing Method Plasmid Transformation Efficiency (cfu/ug)
Slow Freeze PUC19 1*10^7
Fast Freeze PUC19 6.2*10^7

Slow Freeze PCANTAB5E 1.2*10^6
Fast Freeze PCANTAB5E 3.2*10^6


Note that the transformation efficiencies are a little low because in this situation I am transforming into bacteria which already contain a plasmid. The end result are bacteria which contain two plasmids with two different antibiotics. Nevertheless, it should still be possible to compare the different freezing methods since both groups were treated the same with the exception of the freezing method.

So from these results it looks like the fast freeze method is actually a little better. I will admit that there could be one other possibility. The cryogenic tubes I used did not fit flushly/snugly into the tube placeholders in the slow freeze ispropanol cryogenic container. In other words, my tubes my have been a little too small, and therefore may not have dropped in temperature exactly by 1 C per minute. However, I doubt this factor influenced the results much.

#4 Adrian K

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Posted 06 August 2011 - 10:01 PM

Dear azim58,

After I had aliquot my cells, I directly put it into -80 freezer, without ethanol bath or cryofreezing container.
Why not you compare this approach?

IF this approach transformation efficiency really falls between the two approaches you had done, I think you just have enough data to publish a paper. :D
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#5 lab rat

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Posted 06 August 2011 - 10:38 PM

I have also put vials into a -20 C overnight, then transferred to -80 C. The cells worked just fine.
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#6 azim58

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Posted 07 August 2011 - 03:49 PM

@Adrian K

I'll let you know if I test putting the cells directly into the -80 C. I actually hadn't even considered that. However, I don't know if I'll have time to compare multiple freezing methods again. If you test it, I'd be interested in the results though :) .

#7 Adrian K

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Posted 08 August 2011 - 10:39 AM

I did not test the efficiency of the method I mention till this very day. :P
However, I do not encounter any problem in my transformation, yet. At least 30+ transformation to date.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#8 azim58

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Posted 08 August 2011 - 08:35 PM

It's good to know that you can just put the cells directly into the -80 C and get transformable colonies. However, I am more interested in the numbers of how the transformation efficiency directly compares with other methods. For a lot of applications, people just need at least one colony and so several (or even a hundred) colonies will suffice. However, for some applications, people need tons of colonies if they are making a cDNA library or phage library etc.

If I understand you correctly, you might have some numbers soon. . .? Anyway, let me know if you ever compare methods and put some numbers to them.

#9 Adrian K

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Posted 09 August 2011 - 07:36 AM

Okay, I keep you posted if I ever done any efficiency test in the future.
^_^
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#10 azim58

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Posted 09 August 2011 - 01:26 PM

Great I'll do the same.




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