Re: [DIYbio] Anyone working with plant protoplasts?

Looks like you can order directly from that manufacturer/brand-name:
http://www.thomassci.com/Equipment/Centrifuges/_/Hand-Driven-Centrifuge/

On Thu, Jun 20, 2013 at 12:10 AM, Cathal Garvey (Android)
<cathalgarvey@cathalgarvey.me> wrote:
> Awesome, thanks!
>
>
> Sebastian Cocioba <scocioba@gmail.com> wrote:
>>
>> This one is fancy and in your neighborhood, cathal.
>>
>>
>> http://www.djblabcare.co.uk/djb/product/1136/Centrifuges-C1011-Hettich_Hand_Centrifuge
>>
>> Sent from my Windows Phone
>> ________________________________
>> From: Sebastian Cocioba
>> Sent: 6/20/2013 3:02 AM
>> To: diybio@googlegroups.com
>> Subject: RE: [DIYbio] Anyone working with plant protoplasts?
>>
>> http://www.amazon.com/dp/B006OCRE02/ref=cm_sw_em_r_am_wp_am_us?ie=UTF8
>>
>> I think I snagged the last one. Carolina sells a similar device for ten
>> dollars more.
>>
>> Sent from my Windows Phone
>> ________________________________
>> From: Cathal Garvey (Android)
>> Sent: 6/20/2013 2:39 AM
>> To: diybio@googlegroups.com
>> Subject: RE: [DIYbio] Anyone working with plant protoplasts?
>>
>> Link?
>>
>> Sebastian Cocioba <scocioba@gmail.com> wrote:
>>>
>>> I'm gonna try the $70 hand cranked swing out centrifuge from amazon. The
>>> protoplasts need very little g force so maybe this could work. Maybe a
>>> hacked version with a nema stepper? Sure beats 500 bucks worth of equipment.
>>> The low speed should minimize any projectile damage to a mild bruise and
>>> maybe a broken window. Someone reviewed the crank and calculated a 260g
>>> force at 75rpm. It holds 4 15mL tubes. Seems perfect for the job. Ill chime
>>> in on Monday after a ficoll run using the crank. Hopefully this can overcome
>>> one of the many technical hurdles in the quest for perfect 'plasts.
>>>
>>> Sent from my Windows Phone
>>> ________________________________
>>> From: Cathal Garvey (Android)
>>> Sent: 6/19/2013 3:10 PM
>>> To: diybio@googlegroups.com
>>> Subject: Re: [DIYbio] Anyone working with plant protoplasts?
>>>
>>> Yes, layered gradient of different sucrose concentrations. Can be done in
>>> a fixed angle (say, 45 degrees) rotor with slow acceleration/deceleration
>>> but obviously not as well as with swinging buckets, and not at all in a
>>> horizontally fixed rotor like a dremelfuge.
>>>
>>> John Griessen <john@industromatic.com> wrote:
>>>>
>>>> On 06/18/2013 10:07 PM, Nathan McCorkle wrote:
>>>>
>>>>>
>>>>>
>>>>>
>>>>> Probably, it's an air gun firing 1 micron gold particles coated with
>>>>> DNA (add wet DNA solution, then dry, then shoot)
>>>>>
>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>> Sounds like lots of engineering.
>>>>>
>>>>>
>>>>> Maybe not
>>>>>
>>>>>
>>>>>> PEG sounds like another process add-on to my
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>> incubator/mild-centrifuge/liquid-handling/air-PCR-performing/OD-measuring
>>>>>> 15cm x 20cm x 10cm robot idea.
>>>>>
>>>>>
>>>>> Maybe, see below. I would think that with enough energy, you could
>>>>> make both devices
>>>>> and have two separate and complementary products.
>>>>
>>>>
>>>> Sure, and also, by design, the products have many common parts, keeping
>>>> costs down.
>>>>
>>>>>
>>>>>
>>>>>
>>>>> On Tue, Jun 18, 2013 at 5:53 PM, Sebastian Cocioba wrote:
>>>>
>>>> The goal is to
>>>>>>
>>>>>>
>>>>>>
>>>>>> obtain clean, healthy cells and the best way so far is by ficoll or
>>>>>> sucrose gradient centrifugation. The catch is the need for a swing out
>>>>>> centrifuge so the protoplast separate into clear bands at the
>>>>>> interface
>>>>>>
>>>>>>
>>>>>> of a two or three step gradient.
>>>>>
>>>>>
>>>> Can you point me to a clearer picture of this, describe a little more,
>>>> or make a sketch?
>>>>
>>>> Are you saying the media to centrifuge is layers of different densities
>>>> carefully poured into a centrifuge vial?
>>>>
>>>>
>>>>
>>>>
>>>>
>>>> Sebastian Cocioba wrote: Any sucrose
>>>>>>
>>>>>>
>>>>>>
>>>>>> polymer that won't be taken up or is metabolically inert would work.
>>>>>> Just keeping
>>>>>> these guys alive is proving to be the fundamental problem.
>>>>>> Devising a good
>>>>>> protocol would be a great boon for DIY plant bio so I see it as a
>>>>>>
>>>>>>
>>>>>>
>>>>>> worthwhile endeavor.
>>>>>
>>>>>
>>>> Nathan McCorkle wrote:
>>>>>
>>>>> I mention it being similar because during PEG treatment, the cells are
>>>>>
>>>>>
>>>>>
>>>>> quite unstable, so if you jostle them too much or
>>>>> stir the solution
>>>>> too vigorously you simply break the cells open. I've had the same
>>>>> experience with spheroplast preparation for transformations in
>>>>> bacteria (either e.coli or b.subtilis, can't remember), mix too
>>>>>
>>>>>
>>>>>
>>>>> hard/fast and you kill your little buddies.
>>>>
>>>>
>>>> Then maybe a mild slow centrifuge, plus some handler automation
>>>> to prep the stepped gradients is a way to consider?
>>>>
>>>> How could one use stepped gradients without a swing out centrifuge
>>>> holder?
>>>>
>>>>
>>>>
>>>> Use a gel so it could centrifuge sideways?
>>>>
>>>> Swing out is higher danger, higher cost... harder to automate with cheap
>>>> blind robots,
>>>> limits range of uses for a spindle. This kind of task might need a
>>>> dedicated spindle
>>>>
>>>>
>>>>
>>>> with swing out holders.
>>>>
>>>> Still, swing out holders could be low cost if it ran at mild G's and
>>>> took a while.
>>>>
>>>> What time and G's separate protoplasts?
>>>
>>>
>>
>
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--
-Nathan

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