DIY CaCl Transformation

[Cathal Garvey]

Hey all,

I'm fairly convinced that making E.coli stocks before testing in B.subtilis might be prudent until I have a steady supply of plasmid. PCR has to wait for some necessary reagents anyway.

In the meantimes, CaCl transformation: discuss.

I know the story. You grow E.coli cells to a certain OD so that they're still in exponential, then chill them in CaCl and rinse repeatedly by centrifugation and resuspension in CaCl, then add DNA and heatshock/recover, then plate on antibiotic plates.

Facts:

I have a fridge and a dremelfuge.

I don't have a shaking incubator.

My incubator runs at 30 degrees, though I could crank up to 32. I *could* invest a day creating a 37C incubator but I'd rather not.

I don't have a spec or any other way of measuring OD right now.

Questions:

Is the OD actually important, or are you only interested in getting pre-stationary cells? In other words, if I play it safe and harvest cells early, I'll get fewer cells..but will they be just as good?

Are there any reliable ways of growing E.coli in the absence of shaking? Is a shallow stationary flask as good?

How might one calculate differences in growth rate for E.coli at 30C? If it's "grow for 6hr at 37C shaking", is there a chart I could consult to guesstimate the time for "Grow for Xhr at 30C Still"?

Any hot tips for enhancing efficiency?

Will TE buffer interfere with the CaCl method? Is too much CaCl a problem, if I ramped up the CaCl a little to compensate for EDTA content?

Thanks all,

Cathal

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[Cathal Garvey]

Oh yea, and if anyone has a copy of the original paper (Paywall'd http://www.ncbi.nlm.nih.gov/pubmed/383576 ), could I get a copy? I realise it's vastly outdated by now. I just like looking at the older Materials and Methods, I often learn some neat hacks from guys who didn't expect all the technical luxuries we have these days.

[Jordan Miller]

shaking is more important than temperature. they will grow at room temp as long as you shake, nutrient transport and oxygenation are the most important. you can make a growth curve with a red LED (600 nm) and an optical sensor on an arduino. 

jordan

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[Rikke Rasmussen]

If you can wait for about 36 hours, I can send you a copy of the article when I get back to SF from NYC tomorrow afternoon - internet tethering is too slow to do so now.

Also applies to future wishes - I still have full access to the University of Copenhagen subscriptions, and am more than happy to break a few rules to share with others.

/Rikke

On Jul 14, 2011 9:26 AM, "Cathal Garvey" <cathal...@gmail.com> wrote:
Oh yea, and if anyone has a copy of the original paper (Paywall'd http://www.ncbi.nlm.nih.gov/pubmed/383576 ), could I get a copy? I realise it's vastly outdated by now. I just like looking at the older Materials and Methods, I often learn some neat hacks from guys who didn't expect all the technical luxuries we have these days.

On 14 July 2011 14:25, Cathal Garvey <cathal...@gmail.com> wrote:
>
> Hey all,

> I'm fairly con...

[Cathal Garvey]

Thanks Rikke, that's awesome!

Jordan: I understand why shaking helps with aerobic growth, but I never got why it's needed for facultative anaerobic growth such as with E.coli. Can't it simply grow anywhere in the tube, and take nutrients/shed waste by diffusion and by swimming to better places? Even if aeration is needed, wouldn't a wide-bottomed flask and limiting broth to a 2mm depth be OK for aeration?

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[Jordan Miller]

attached.

jordan

1. Dagert M, Ehrlich SD. Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene 1979 May;6(1):23–28.

[Cathal Garvey]

Thanks Jordan! I'm seeing a lot of one-step methods involving PEG, which are attractive, but I'm trying to determine whether or not DMSO is a requirement or just a convenience for freezing in those cases.

It's always great to have the original papers..

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[Jordan Miller]

dunno, guess we gotta try it! i think you might get a bunch of dead e. coli cells if you grow statically, which can harm protein expression yields. Also, diffusion doesn't always work, it's possible you might get a stable waste gradient with bacteria in a toxic phase environment (waste byproducts may form a phase transition).

i would strongly guess shaking at room temperature will give you much better growth curves than static at 37 ºC.

wide bottom flask should work fine, hadn't thought of that as an option. it's just so space inefficient. but if you have plenty of space and no shaker, try it!

jordan

[Cathal Garvey]

Well I know they'll grow, but I never bothered checking what proportion of the final mix is living/dead/dormant! :) I've made DH10B grow just by propping a 30ml LB tube against the side wall of my incubator (which, to remind you, is just a polystyrene box with a thermostat and pet heater). So they're pretty tough, but transformation efficiency might suck; it's entirely possible that, as you suggest, a gradient appears and cells below the surface hit stationary way sooner.

[Jonathan Street]

Can you not cobble something together to shake one flask?  That box you were using was definitely big enough to suspend a flask in.  Then you just need a small motor with a off-centre mass attached to the shaft.

[Rikke Rasmussen]

If you've got an old cell phone with a buzzer lying around, you can extract the motor - it'll have an off-set on the shaft and should be powerful enough to use as a shaker.

/Rikke

[Cathal Garvey]

This paper is excellent on the one-step method using PEG:

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

They go into buffer design, which is great. They discuss the efficiency of different combinations of PEG/DMSO/Cation, and which cations they used etc.

Ultimately it looks like you could use just MgSO4 (Epsom Salt) and PEG-3350 (Miralax in US, various other laxatives in EU) to get a decent one-step transformation by resuspending early-growth E.coli in a MgSO4+PEG buffer, adding plasmid, and leaving on ice. Heat shock optional.

On 14 July 2011 14:53, Cathal Garvey <cathal...@gmail.com> wrote:

[General Oya]

Cathal,
If you have an old turntable it could be used as a makeshift shaker, just control the room temperature.

Ryan

[Cathal Garvey]

I could easily fit a turntable into my incubator.. but I don't have one. It's an idea though to get one and hack it to jitter back and forth; they often have rubber grips already installed, so that'd be a pretty easy hack with an arduino I think.

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[General Oya]

A small children's 45 player sounds ideal, maybe a junk shop. Any Goodwill type shops on the emerald isle?
Ryan

[Cathal Garvey]

Plenty! But record players wouldn't have been so common that they turn up all the time. Still, worth a look, definitely!

[ruphos]

On Thu, Jul 14, 2011 at 7:13 AM, Cathal Garvey <cathal...@gmail.com> wrote:

This paper is excellent on the one-step method using PEG:

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

This link seems to be broken. Do you have a DOI or PubMed link?

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-- Friedrich Nietzsche

[Derek]

What a timely thread! I'm putting together an intro transformation
class at Biocurious this summer and have just ordered the reagents for
a CaCl approach. For the past couple of years I have been using the
CCMB80 approach from http://openwetware.org/wiki/TOP10_chemically_competent_cells

At a fully equipped university lab I was getting better transformation
rates from that, but I think the CCMB80 approach is more sensitive to
temperature and agitation than the CaCl approach (at least it failed
reasonably often when I didn't follow the protocol exactly whereas the
CaCl approach just had lower transformation efficiencies when I got
sloppy.)

I'll post pictures and protocol when I get it all set up down here in
a couple weeks...

--Derek

On Jul 14, 10:10 am, ruphos <apokrup...@gmail.com> wrote:

[Rikke Rasmussen]

We've just done exactly that at Genspace. Their protocol is from www.carolina.com, so you might be able to grab and modify that?

On Jul 14, 2011 2:55 PM, "Derek" <der...@gmail.com> wrote:
What a timely thread! I'm putting together an intro transformation
class at Biocurious this summer and have just ordered the reagents for
a CaCl approach. For the past couple of years I have been using the
CCMB80 approach from http://openwetware.org/wiki/TOP10_chemically_competent_cells

At a fully equipped university lab I was getting better transformation
rates from that, but I think the CCMB80 approach is more sensitive to
temperature and agitation than the CaCl approach (at least it failed
reasonably often when I didn't follow the protocol exactly whereas the
CaCl approach just had lower transformation efficiencies when I got
sloppy.)

I'll post pictures and protocol when I get it all set up down here in
a couple weeks...

--Derek

On Jul 14, 10:10 am, ruphos <apokrup...@gmail.com> wrote:
> On Thu, Jul 14, 2011 at 7:13 AM, Cathal Garvey <cathalgar...@gmail.com>wrote:

>
> > This paper is excellent on the one-step method using PEG:

> >http://www.pnas.org/content/86/7/...

[Bryan Bishop]

I don't think the Genspace transformation was CCM80.

[Rikke Rasmussen]

No, it was CaCl - that was the point.

[Meredith L. Patterson]

Someone made a shaker from LEGO for PCB etching; this looks promising.

http://www.youtube.com/watch?v=h1gzOHE5Uh8

--mlp

[Cathal Garvey]

I strongly recommend the PEG-MG-DMSO method after reading about it. It is so much easier. There's a protocol on OWW ans I'll share the paper tomorrow.

[General Oya]

We were getting better transformation rates from CaCl then the Competent cells we picked up from NEB. But in both cases we had to be a good 24 hours behind what we had expected before results really became visible.

Ryan

[Nathan McCorkle]

If you're not transforming a library, you don't need high efficiency.
I've done an overnight culture, then in the morning subcultured it and
a few hours later when it got turbid I used that for transforming.
Rinse twice with 50mM CaCl2, add DNA, heatshock for 45-90 seconds,
recover with LB or SOC (latter preferred). Keep things as icy-cold as
possible before the heat-shock, let the DNA chill with the cells for
5-20 minutes before heatshocking.

For good measure I've thrown all glass/plasticware and solutions into
the freezer about an hour before doing the transformation (tubes,
tips, anything that touches or goes into the reaction)

Check out the research I did and the documents I compiled for the last
undergrad Molecular Bio lab that I taught in the DIYbio discussion
"I'd like to discuss transformation methods (bacterial)"

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[General Oya]

Thinking about it earlier, I wondered whether you might just tape a tube to a metronome and go.

Ryan

[Nathan McCorkle]

On Sat, Jul 16, 2011 at 10:20 PM, General Oya <gener...@gmail.com> wrote:
> Thinking about it earlier, I wondered whether you might just tape a tube to
> a metronome and go.
>

huh?

[General Oya]

In reference to low cost shaker for e.coli

[Patrik]

I bet that would work too. Although a second hand metronome may be
harder to find, more expensive, and less powerful than a second hand
turn table. If you prop the turntable up at a bit of angle, and
distribute the load evenly, you should be able to stack quite a few
tubes or petri dishes on it.

On Jul 16, 10:20 pm, General Oya <general...@gmail.com> wrote:
> Thinking about it earlier, I wondered whether you might just tape a tube to
> a metronome and go.
>

[Cathal Garvey]

I can personally attest that the PEG3350/MgSO4 (Laxative/Lubricant and Epsom Salt) method of transforming E.coli works like a charm. Easiest transformation method I've ever tried, an absolute no-brainer. And, as it doesn't require a centrifuge, it's probably the best DIYbio method out there.

Attached is the paper. You can and should autoclave the TSS medium to sterilise it, but if you're using DMSO (as recommended), you might want to add that afterwards. It'll be sterile anyway if it's neat DMSO.

Brand names for PEG-3350, which are mostly laxatives, include: Carbowax, GoLYTELY, GlycoLax, Fortrans, TriLyte, Colyte, Halflytely, Macrogol, MiraLAX, MoviPrep

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[Cathal Garvey]

Oh, and just to add:

I didn't agitate the cells. I was too impatient to get stuff done short-term, so I just went ahead and grew a starter culture in a test tube standing overnight, then used 1ml of that culture to start a 100ml conical flask. I took cells from that conical flask about 3hrs later and used them as my early-mid exponential cells. I *did* shake the flask occasionally, though. :)

All culturing was done at 30C.

So, minimal infrastructure for transforming E.coli:

- Terrible incubator
- Pressure Cooker

- Mid-Low volume glassware

- Pasteur Pipettes

- Plasmid Free E.coli (I used DH10B)

- Off-the-shelf PEG-3350 Laxative

- Off-the-shelf MgSO4 / Epsom Salt

- Deionised Water

- LB medium or ingredients to make LB medium

--- Namely: Soy or milk protein, Yeast Extract, Salt, Bromelain/Papain

- A plasmid to transform with

- If required, antibiotic to select for transformants

- Ice

However, I didn't use minimal: I used a centrifuge and resuspended 10x concentrated cells in 1x transformation medium, and got great results. I'd expect poorer results if using the minimal method, but according to the paper you can totally just combine fresh early-exponential cells and 2x TSS and expect transformants.

[Nathan McCorkle]

Very cool... good paper too, I might check out the Klebe paper they
referenced, as well as the CaCl2 Mandel and Higa... just for reference
:)

If I get to teach undergrad molecular bio again this year, I might
include this and a cheap electroporation tech that's been discussed
here, along with the standard CaCl2 protocol that is part of the
coursework.

Also, the paper mentions CaCl2 transformation efficiency decreasing
from 10mM to 100mM, so you could probably substitute CaCl2 for Mg2+ if
its easier to come by (I think rock salt is CaCl2, but maybe epsom
salts are purer)

--

[Cathal Garvey]

My guess is that pure Epsom is easier to come by, but I'm not sure about Rock Salt; good lead on cheap CaCl if so! :)

The only question mark with MgSO4 is the hydration level, which makes a big difference to the molar weight. Chances are you'll be buying the heptahydrate, but the anhydrate is used sometimes for drying purposes (it's really hygroscopic). I think the heptahydrate is glassier in appearance and the anhydrate is more free-flowing and powdery...but having never actually *seen* the anhydrate (and being too lazy to make some) I can't say for sure.

The convenient thing is that the precise molarity doesn't matter according to the paper provided its between 20-50 mMol, so when writing up instructions I gave a compromise value for how much Epsom to add. It ends up being either close to 50mMol if you're using the anhydrous form, or 20mMol if you're using the Heptahydrate. That helps avoid some confusion.

[Cory Tobin]

> My guess is that pure Epsom is easier to come by, but I'm not sure about
> Rock Salt; good lead on cheap CaCl if so! :)

Rock salt is just NaCl but in an impure, mineral form. I wouldn't use
it in any solutions. Too many impurities. I thought the mineral went
by the name "Holite" but wikipedia turns up nothing for holite - must
have remembered wrong.

> really hygroscopic). I think the heptahydrate is glassier in appearance and
> the anhydrate is more free-flowing and powdery...but having never actually
> *seen* the anhydrate (and being too lazy to make some) I can't say for sure.

Anhydrous looks like powdered sugar, except less soft. Kind of
chalky. The stuff you get at the grocery store is definitely the
hydrate.

Also, have you tried freezing the TSS competent cells? I'm curious to
know how well the cells frozen in TSS transform after thawing.

-cory

[Cathal Garvey]

If you make TSS with DMSO, they do (according to the paper). I doubt they would without the DMSO: PEG3350 is wax at RT, so I don't imagine it'll act as a decent cryoprotectant (but perhaps all that's important is the effect it has on crystal formation?).

Then again, I can't test without a -80. I could try -20 another time and let ye know, but for now I can only speak to instantaneous transformation efficiency.

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[Nathan McCorkle]

On Fri, Jul 22, 2011 at 1:35 AM, Cory Tobin <cory....@gmail.com> wrote:
>> My guess is that pure Epsom is easier to come by, but I'm not sure about
>> Rock Salt; good lead on cheap CaCl if so! :)
>
> Rock salt is just NaCl but in an impure, mineral form.  I wouldn't use
> it in any solutions.  Too many impurities.  I thought the mineral went
> by the name "Holite" but wikipedia turns up nothing for holite - must
> have remembered wrong.

You mean Halite, which is NaCl... I meant Driveway Melter... which is
CaCl. Searching Amazon for "calcium chloride" yields driveway melter,
as well as food grade, and aquatic grade CaCl

>
>
>> really hygroscopic). I think the heptahydrate is glassier in appearance and
>> the anhydrate is more free-flowing and powdery...but having never actually
>> *seen* the anhydrate (and being too lazy to make some) I can't say for sure.
>
> Anhydrous looks like powdered sugar, except less soft.  Kind of
> chalky.  The stuff you get at the grocery store is definitely the
> hydrate.
>
> Also, have you tried freezing the TSS competent cells?  I'm curious to
> know how well the cells frozen in TSS transform after thawing.
>
>
> -cory
>

[Mega]

When doing the PEG transformation with laxatives, how do you sterilize it? Just in the pressure cooker? or will the PEG dissociate?
Filter sterilization?

[Nathan McCorkle]

You probably just rely on your genetic selection marker after
transformation. Most google stuff says filter sterilize.

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[Mega]

You're sure about that?

Note: Ok, (most) bacteria will die. funghi will happily survive ampicillin, so if I get spores inside??

[Avery louie]

IIRC, peg+MgSO4 can be autoclaved, but not the DMSO, which is perma-sterile.  I will see if I can find a reference

--A

On Fri, Aug 10, 2012 at 9:26 AM, Mega <masters...@gmail.com> wrote:

You're sure about that?

Note: Ok, (most) bacteria will die. funghi will happily survive ampicillin, so if I get spores inside??

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[Avery louie]

http://www.bio.net/bionet/mm/methods/1994-February/011521.html

http://www.koko.gov.my/CocoaBioTech/DNA%20Cells32.html#solutions

consensus seems to be to clave, not to not clave.

also, if you do filter sterilize DMSO, be sure your filter can handle it.  Mine melted my filter, and then maybe some of the bottle, until the bottle-bits started to crystallize out of the DMSO.  It was alarming.

--A

[Nathan McCorkle]

DMSO is a great way to get small molecules through your skin too, owing to its solvent properties. If its capable of melting a filter, get a better (resistant) filter or don't filter it.... the melted plastic will go into your reaction and could throw a wrench into the project's 'gears'

[Jeswin]

On Fri, Aug 10, 2012 at 1:31 PM, Nathan McCorkle <nmz...@gmail.com> wrote:
> its solvent properties. If its capable of melting a filter, get a better
> (resistant) filter or don't filter it.... the melted plastic will go into

I think you're supposed to use nylon membrane filters.

[Cathal Garvey]

Technically you don't need the DMSO at all. It does improve
transformation efficiency, but the main reason for DMSO in the TSS
medium was so that competent cells could be stored in -80 freezers ready
for use.

I've done transformations several times with only Peg/Mg and it works
great. You can indeed autoclave-sterilise it, although shake before use;
heavy PEG seems to settle out a little over time.

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