You can get away without shaking, *if* the cultures are grown in shallow
broth. You should still give them an occasional swirl though.
And yes, they should be warm. I have successfully transformed E.coli
grown at 30C. I incubate using a terrarium heater mat, a pet-shop
thermostat, and a polystyrene box. I use an external thermometer to
sanity-check the temperature, because commercial pet thermostats are
rarely correct (but very stable, I find).
If using heater wire to heat your terrarium, be careful to insulate the
wire somehow to keep it from melting/igniting the polystyrene!
On 31/01/12 09:16, Nathan McCorkle wrote:
> they really should be warm and shaking before transformation... try
> using a cell-phone vibrator (or playstation/nintendo controller
> vibrator) attached to an eppendorf tube for shaking/aeration on the
> cheap
>
> On Tue, Jan 31, 2012 at 4:07 AM, Mega <masterstorm123@gmail.com> wrote:
>> Next question:
>>
>>
>> To transform bacteria they have to be in a state of exponential
>> growth. Is this really imperative or just increases transformation
>> efficiency??
>>
>>
>> ____
>> For my transformation I think about building a breeding box using an
>> ATtiny, LM335, a box of polystyrol and some wire.
>> But in case this fails, would transformation still work?
>>
>>
>>
>>
>> On 27 Jan., 17:15, Mega <masterstorm...@gmail.com> wrote:
>>> "Clearly, I wouldn't be so strict with DNA that was proven to have no
>>> ecological consequences; no resistance genes, no ecological-unknowns.
>>> A
>>> plasmid containing only GFP and plasmid maintenance genes is hardly
>>> worth worrying about, for example. "
>>>
>>> I totally agree with that...
>>>
>>> By the way, the gfp could not be taken up and expressed by plant
>>> seemen, because the (bacterial) promotor wouldn't be readable?? It
>>> would be junk - DNA?
>>>
>>> On 27 Jan., 12:33, Cathal Garvey <cathalgar...@gmail.com> wrote:
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>> Actually, leaving the cells to "overcook" will usually destroy all
>>>> antibiotics present; it's only unused media that really needs boiling or
>>>> other such treatment before disposal. In the case of chloramphenicol,
>>>> I'll probably just add some resistant cells to unused media and incubate
>>>> in order to destroy the broth, then boil-kill and UV inactivate.
>>>
>>>> Microwaves don't explicitly damage DNA.
>>>> However, UV does, and I would advocate using UV to mince DNA before
>>>> disposal, especially if the DNA contains medically significant (i.e.
>>>> AmpR, maybe Chlor?) antibiotic resistance genes. Most labs have UV
>>>> transilluminators for Gel electrophoresis, even though blue illumination
>>>> is more all-round useful (precisely because it doesn't mince DNA).
>>>
>>>> So, if I were working with E.coli, which can't survive boiling, I'd
>>>> simply boil cellular waste in a glass beaker or flask, then put the
>>>> container on a UV illuminator for 5 minutes before disposal.
>>>
>>>> If I were working with B.subtilis, I'd autoclave rather than boiling,
>>>> and do the same.
>>>
>>>> Clearly, I wouldn't be so strict with DNA that was proven to have no
>>>> ecological consequences; no resistance genes, no ecological-unknowns. A
>>>> plasmid containing only GFP and plasmid maintenance genes is hardly
>>>> worth worrying about, for example.
>>>
>>>> Of course, opinions differ, and we've had animated discussions here in
>>>> the past on this issue; whether to bother destroying antibiotics,
>>>> whether to bother destroying DNA.
>>>
>>>> On 27/01/12 08:54, Mega wrote:
>>>
>>>>> Ah, you only want to dispose of the plasmids left from transformation?
>>>
>>>>> On 27 Jan., 09:53, Mega <masterstorm...@gmail.com> wrote:
>>>>>> Have you considered using UV-radiation? I saw one on a TV 'show-
>>>>>> documentation-infotainment' which costed about 20 bucks and they
>>>>>> tested if it really worked.
>>>
>>>>>> And, surprisingly, it really worked fine!
>>>
>>>>>> Maybe Microwave sterilization could also work when it damages the DNA
>>>>>> ( not only heat up the bacteria). Or you add e.g. ampicillin to kill
>>>>>> the bacteria and then heat the whole pot to destroy the amp.
>>>
>>>>>> On 26 Jan., 23:17, Cathal Garvey <cathalgar...@gmail.com> wrote:
>>>
>>>>>>> Actually, a control Bacillus plasmid I'm going to be using is Chloramphenicol resistant: I hadn't realised it was heat resistant! I'd better imagine up a disposal procedure..
>>>
>>>>>>> Venkatesh Srinivas <m...@endeavour.zapto.org> wrote:
>>>>>>>> On Wed, Jan 25, 2012 at 11:05 PM, Tom Randall <tarand...@gmail.com>
>>>>>>>> wrote:
>>>>>>>>> ...
>>>>>>>>> I am sure there are others. No environmental issues with amp or
>>>>>>>> others
>>>>>>>>> if you autoclave everything before you dispose of it, this will
>>>>>>>>> inactivate most antibiotics that I am aware of. If there are any that
>>>>>>>>> can withstand autoclaving I would be interested in knowing. This is
>>>>>>>>> why one normally adds antibiotics to media after autoclaving the
>>>>>>>> media
>>>>>>>>> and cooling to ~55-60C to avoid their inactivation.
>>>>>>>>> Go wild!
>>>>>>>>> ...
>>>
>>>>>>>> I believe chloramphenicol is thermostable; should survive autoclaving.
>>>>>>>> Not commonly used in DIYbio I imagine though...
>>>
>>>>>>>> --vs;
>>>
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>>>
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>>>
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