RE: [DIYbio] Anyone working with plant protoplasts?

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

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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?