Re: [DIYbio] Miniprep without EDTA

> 6-8 hours also when 20°C room temperature??
> Won't they need more time as grown-at-37°C-ones?

No, I culture at 30C, which is still nice and warm. I do leave them
longer though, maybe 10 hours. When you get used to them, you can often
tell by the turbidity (cloudiness) and dispersion of a culture roughly
how "happy" it is. Exponential cells seem, to me, to "clear" less when
allowed to settle after a shake, probably because they're still actively
swimming around. On the other hand, stationary cells seem to me to be
more easily stratified by resting on a bench.

> Interesting. So natural plasmids are far more stable? A professor of mine
> always tells us that bacteria don't have luxury and so lose their plasmids
> kind-of-always.
> I don't know if pVIB has a pUC origin or something similar. The Carolina
> homepage doesn't say.

Not so! Plasmids wouldn't even exist in the wild if they were that unstable.

Basically, most of our plasmids use a mechanism called "Rolling circle
replication" to make copies of themselves. This mechanism involves a
step where the plasmids are single-stranded DNA, a very unstable state
for DNA to be in.

In wild plasmids, there's a non-coding region (often called a "Single
stranded origin" or SSO, but names differ) that folds into a special
shape that encourages transcription into mRNA by host ribosomes, but
only for 5-20 nucleotides or so. This mRNA acts as a "primer" for DNA
polymerases to replicate the other strand of the plasmid, stabilising it.

However, when we first started "hacking" wild plasmids to make early
cloning vectors, we just hammered in antibiotic resistance sites to make
manipulation easier, and then started cutting away whatever we could
remove while still having a functional plasmid. With
antibiotic-resistance genes and antibiotics forcing the cells to retain
even highly unstable plasmids, many or most cloning vectors of the time
ended up having their SSO's cut out, and because very unstable. Because
most modern vectors are simply re-hashes of older vectors with fancy new
features, they are similarly unstable.

This instability is one of the things I aimed to target with my own
plasmid, by deliberately including a highly stable SSO close to the
"normal" origin and Rep genes. The available literature suggests that
unless a gene is particularly bad for a bacterium (toxic gene products,
overly strong promoters, etc..), the plasmid shouldn't get lost just due
to growth and replication. You get less than 2% of cells without
plasmids after ~10-20 cycles of replication without antibiotic
selection, versus 80-99% for conventional plasmids.

I don't yet know if my plasmid matches this stability. That's one of my
remaining questions to answer! ;)

On 07/06/12 16:18, Andreas Sturm wrote:
>> 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.
>
> Interesting. So natural plasmids are far more stable? A professor of mine
> always tells us that bacteria don't have luxury and so lose their plasmids
> kind-of-always.
> I don't know if pVIB has a pUC origin or something similar. The Carolina
> homepage doesn't say.
>
>
>> 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.
>
> 6-8 hours also when 20°C room temperature??
> Won't they need more time as grown-at-37°C-ones?
>
>
>
>
>
> 2012/6/7 Cathal Garvey <cathalgarvey@gmail.com>
>
>>> 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.
>>
>> --
>> www.indiebiotech.com
>> twitter.com/onetruecathal
>> joindiaspora.com/u/cathalgarvey
>> PGP Public Key: http://bit.ly/CathalGKey
>>
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>


--
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