Re: [DIYbio] Miniprep without EDTA

> I recently got a Dremelfuge from a friend who built his own 3D printer. As
> an inauguration present ;)
> 1) *Will it be spinning fast enough to do a miniprep* (Get most of the
> bacteria out of soultion)?? I once tried it, but it gave very little
> yield. But maybe, the lb marmelade jar was not full of of bacteria
> (althought they didn't glow anymore, so lysis?)

That depends. As always, and especially because you printed your own, I
can't offer any suggestions or guarantees of safety. But, I find that
5-10 minutes on the second setting of a Dremel 300 is enough to pellet
bacteria, although if you're in a hurry you can do it faster at setting
3-5. Remember that the longer/harder you spin them, the harder the
pellet will be to resuspend, and the higher the chances are that you'll
kill/damage whatever you're trying to pellet/resuspend.

But yes; Dremelfuges are easily able to pellet bacteria.

> 2) *In which container shall I breed the 3-5 mL LB solution for the
> bacteria??
> *They will need oxygen, so above the tube shall be space. I have e.g. those
> https://encrypted-tbn0.google.com/images?q=tbn:ANd9GcQ8Q34n7YkKUL1gx99j4OL3efA0_NN9AgO2BHv28QgmxIfdT0w8(the
> right one, 15 mL)
>
> Is that sufficient?

A wide & shallow-bottomed container is perfect if you have a jar or
erlenmeyer flask, but if you can shake them then a test tube or similar
is fine too. If you're using a deep container like a test tube, and you
don't have a shaking incubator, try to keep the volumes low and keep the
tubes slanted so that there's maximum air-liquid exposure.

> 3) *Do they need 37°C and shaking?* I don't have a device that heats or
> shakes them. But I have a hacked play-station controller which shakes when
> I apply a voltage. I could tape it to the controller and let it shake
> (never tested it for more than 1/2 an hour, but I'm confiden't it won't
> blow up)
> *How will I know they are ready for being harvested?

If you have access to 37C and shaking, that's perfect. But, I grow my
E.coli at 30C without shaking, and I still get good yields on a
miniprep. You just need to make sure they have plenty of air-broth
contact by inclining them or by only incubating in erlenmeyer flasks
with ~3mm of liquid at the bottom.

Vibration, as from a controller (awesome that you're using a PS
controller! :P), will certainly help to aerate the cultures by agitating
the water (preventing stratification of high-medium-low oxygen fluid)
and creating more surface area due to standing waves. So yea, vibration
is probably good. But do test the motor for overheating; vibrator motors
in phones and games consoles aren't usually designed for extended use.
There is a commonly available sort of vibrator motor that *is* designed
for extended use though.....that's right, massage wands! :D Glad we're
all on the same page!

> 4) Shall I get some ampicillin for that? When they grow at 37°C, they will
> grow faster thus losing plasmids more poften. When I keep them at room
> temperature, they will lose much less plasmids I read. The yield may
> decrease, but will it be significantly?

If you're using a conventional cloning plasmid, you'll need to apply
selection constantly, as they are usually missing DNA regions critical
for stable reproduction in wild plasmids.

For good yields, I recommend "rinsing" the cells you will be inoculating
with in plain broth first. I've seen this recommended by others as a way
to remove any beta-lactamase enzyme that they've secreted into their old
medium; the enzyme genes may be dormant by the time you harvest them for
inoculation of another culture, but the enzymes that remain can quickly
destroy any ampicillin in your new culture, reducing stability and
plasmid yield.

So, suspend the cells in new LB buffer, gently vortex or mix, and
centrifuge back out of that buffer before putting them into your new
Ampicillin-LB for maximum yields.

You don't need to do this for routine stuff, like subculturing a strain
just to keep it stable. It's just a good idea if you want high plasmid
yield, which I think you want.

If you *really* want good yields, you could consider using a different
broth: LB is actually very poor for plasmid yield. TB ("Terrific Broth")
is a common substitute using similar ingredients, which gives better
yields (I am told).

Most basic thing to bear in mind: don't use old cells. Use them while
they're growing happily in exponential-phase. Because you're using pVIB,
this is easy: harvest them for a miniprep when they are glowing at their
brightest, or just after their peak brightness. That indicates lots of
cells in exponential phase, which is just what you want. The moment you
let them fall into stationary phase (old) growth, your yields will drop
significantly.

Good rule of thumb: Harvest 6-8 hours after starting the culture. If
you're doing it overnight, start incubating them late and miniprep the
very next morning.

--
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