Back to Transformation! (Was Re: [DIYbio] Re: Which microscope?)

Hey, awesome to hear that you're covered for a location! That's a really
helpful Prof you've got there. See, academic spirit isn't dead yet!

For your time-sensitive experiment, don't risk a failed sterilisation on
a microwave you haven't used previously for this task. I'm enthusiastic
about microwaves being an effective sterilisation tool, but I'm still
not confident enough of their reliability to recommend them when an
autoclave is at hand!

So, autoclave, then wait until it's cooled enough that you can handle it
without pain yet it's still liquid. That's a good time to add Amp; do it
under sterile conditions, such as close to a blue bunsen flame or under
a HEPA-filtered airflow. Don't shake to mix; cooled molten agar forms
very stable bubbles! Swirl patiently to mix. You're aiming for 100ug/ml
Ampicillin, so for 50mls you want 5mg of Amp.

It's normal to make stock solutions of ampicillin at 1000x (100mg/ml)
strength, and filter sterilise them, but if you can't afford the filters
you'll get away without sterilising the Ampicillin stock. After all,
it's a strong antibiotic! Just don't expect the resulting agar plates to
last forever if you can't sterilise the Amp; make them up fresh and use
within a week or two.

On 01/03/12 10:27, Mega wrote:
> I asked the proffessor if I could have access to the lab to do a
> transformation. And guess what.
>
> Monday I'm doing it.
>
>
> But there are still a few questions...
>
>
>
> When I make an LB Amp Agar Dish, I first mix water and LB Agar. Then I
> heat it up. I think making 50 ml will be enough for one or two petri
> dish.
> Then I have to sterilize it. Will 3 mins in the microwave do the job,
> or will I need to put it into the autoclaveur for 20mins as
> recommended?
>
> And, at <55°C I have to add the ampicillin which is not heat
> ressistant.
>
>

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