Easiest transformation

[Mega]

Good news, everyone.

A proffessor of mine told me that some microbiologists were laughing at them because they used competent e.coli.

Those microbiologists just incubated the colis with the plasmid for an half hour and then selected. There always were some colonies...

of course, this won't work when doing ligation, but for amplyfying a plasmid it is said to do its job...

[Cathal Garvey]

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Did they mention whether this works with any strain, or some
particularly domesticated strains in their labs?

Cool to know this is a "routine" hack, though! I do love timesaving
shortcuts. :)

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[Mega]

I can ask him, when I see him next time. 

Next lecture would be tomorrow, however, I am at the AEC to transform agrobacterium with an empty pGreen vector (that carries KanR and firefly luciferase).

So, maybe within the  next couple of days... 

[Josiah Zayner]

Most competent cells are just cells in a solution of counter ions to help alleviate the the charge on DNA passing through a membrane.

I would assume their transformation efficiency is very very low. And the amount of time and effort saved would be expended twofold by needing to incubate the cells with plasmid for such a long period of time and colony PCRing/sequencing many colonies to find a hit.

[Mega]

Yeah, for ligation it won't be worth surely. but for demonstrating gfp, why not...

I asked him, he said those were just some routine colis. DM109 or something thelike.

[Josiah Zayner]

I don't think there is a bacterial strain called DM109. Perhaps are you referring to JM109? Further, JM109 is used specifically for transformation and plasmid DNA isolation and has many genetic manipulations for such.

Also, I would say if you are expressing GFP most of these plasmids require a T7 polymerase which "normal" E. coli dooesn't have. 

I am sure as you have said these things can work. But why would anyone even try when it takes just as much effort to do it the "correct" way and perhaps even less effort in the long run due to lack of needing to retransform because poor transformation efficiency.

Take some cells add some 5% PEG and a little Mg or Ca and voila your transformation efficiency increases by many orders of magnitude. I would dissuade people from doing as you said unless they are stranded in a Jungle with no access to any mono or divalent cations.

http://www.pnas.org/content/86/7/2172.full.pdf

On Thu, Apr 4, 2013 at 8:52 AM, Mega <masters...@gmail.com> wrote:

Yeah, for ligation it won't be worth surely. but for demonstrating gfp, why not...



I asked him, he said those were just some routine colis. DM109 or something thelike.

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[Mega]

Ok true. 

He said that he didn't know which strain exactly, but he believed XY (two letters) and 109 or 108

Perhaps JM as you said.

On Tuesday, April 2, 2013 3:17:41 PM UTC+2, Mega wrote:

[Nathan McCorkle]

On Thu, Apr 4, 2013 at 7:28 AM, Josiah Zayner <josiah...@gmail.com> wrote:

 I would dissuade people from doing as you said unless they are stranded in a Jungle with no access to any mono or divalent cations.

Paratrooper jungle biotech is one of my startup ideas, actually :P

--
-Nathan

[Mega]

Hi. Today I'll try it with E.Coli JM109. just incubate with plasmid in LB and one in plasmid and water. half an hour incubation duration.

[Josiah Zayner]

Incubating bacteria in water will kill it due to osmotic stress. they will explode.

On Thu, Apr 11, 2013 at 4:22 AM, Mega <masters...@gmail.com> wrote:

Hi. Today I'll try it with E.Coli JM109. just incubate with plasmid in LB and one in plasmid and water. half an hour incubation duration.

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[Koeng]

Lets use that instead of lysis buffer xD

Boom boom boom!

[Mega]

On Thursday, April 11, 2013 3:40:47 PM UTC+2, Josiah Zayner wrote:

Incubating bacteria in water will kill it due to osmotic stress. they will explode.

No, surely not. I once suspended a colony of E.Coli carrying pVIB plasmid in deionized (!) water (in an Eppi) and plated it on agar. There was a bacterial lawn. I think that is a myth. 

[Mega]

By the way... 

 They are in the incubator... 

One in H2O with 0,9% NaCl 37°C, LB 37°C and one in LB 20°C... 

Just inocculated E.Coli with a pipette tip into a sterile eppendorf tube, with 200 uL medium/water. Waited for 30 Minutes and plated them on LB-Apmicillin-Agar

Seems google doesn't want me to upload pictures :/ 

[Josiah Zayner]

Hahaha. It is science! It is not a myth. 

Hypoosmotic stress has been a well observed phenomenon for a long time. If you don't believe me grab a microscope and view it. Personal anecdotes are not science. Who knows why the bacteria survived? 

http://en.wikipedia.org/wiki/Osmotic_shock

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[Mega]

I agree and disagree. 

Obviously, I had something growing on the plates and gloewing in the dark. Maybe osmotic stress killed 70% of the cells. I don't know. But obviously, if it had killed the bugs,my agar plates wouldn't have glowed in the dark.... 

[Koeng]

So how do the transformation work out???????

[Cathal Garvey]

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Bacterial cells are pretty resistant to osmotic shock; they have
strong cell walls for precisely this reason. In fact, due to their
tiny size, bacteria have to accumulate a very hypertonic cytoplasm, so
they are *always* under "osmotic shock", in a sense.

(Plant cells are also generally in a state of mild hyperosmosis, so
they maintain "turgidity" against their cell walls. If you put them in
hypertonic medium, they tend to detach from their walls, but they can
tolerate hypotonic solutions fairly well.)

Of course, incubated for too long in water, they'll die for other
reasons; diffusion of nutrients outside the cell and expenditure of
energy to pump it back in, and starvation.

If cells have been treated with lysozyme, then they'll explode pretty
quickly unless they're in an isotonic buffer, that's why such buffers
are so common in plant protoplastic protocols that use cellulases.

On 04/11/2013 03:05 PM, Josiah Zayner wrote:
> Hahaha. It is science! It is not a myth. Hypoosmotic stress has
> been a well observed phenomenon for a long time. If you don't
> believe me grab a microscope and view it. Personal anecdotes are
> not science. Who knows why the bacteria survived?
>
> http://en.wikipedia.org/wiki/Osmotic_shock
>
>
> On Thu, Apr 11, 2013 at 8:54 AM, Mega <masters...@gmail.com
> <mailto:masters...@gmail.com>> wrote:
>
>
>
> On Thursday, April 11, 2013 3:40:47 PM UTC+2, Josiah Zayner wrote:
>
> Incubating bacteria in water will kill it due to osmotic stress.
> they will explode.
>
>
> No, surely not. I once suspended a colony of E.Coli carrying pVIB
> plasmid in deionized (!) water (in an Eppi) and plated it on agar.
> There was a bacterial lawn. I think that is a myth.
>
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[Mega]

On Thursday, April 11, 2013 4:52:26 PM UTC+2, Koeng wrote:

So how do the transformation work out???????

Will see it tommorow, when I open the incubator and colonies have grown that show GFP fluorescence...  

[Josiah Zayner]

This is true. My bad. Bacteria won't die from the swelling but starvation or leakage. 

Most other cell types die from the osmotic pressure. My apologies.

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[Mega]

Well, no problem... ;) 

Yeah, just like the plant protoplasts I did.

 Don't know what happened to them, but maybe they were very sensitive and there was not enough Xylitol in the media to mitigate osmotic stress. 

Althought did weigh in as much Xylitol as Mannose would have been required...

[Mega]

Holy crap, seems the transformation efficiency was like 500,000 % ^^  :D 

You can see the bacterial lawn growing there,,, 

The Amp-Plates are some months old, it seems the antibiotic got powerless... 

On one plate there was an  *agaricus* growing, so I cut it out in a sterile manner...

[Mega]

I plated the bacteriea from the 37°C LB plate onto a fresh LB-Amp-plate. Put it into the incubator, will have a look at it after the weekend...