Re: [DIYbio] Protoplast isolation succesful ;)

Images 3 and 4 look like they /could/ be protoplasts?
I think it's usually best to digest cells to protoplasts right before
use, then transform and plate on "recovery" medium, though the precise
steps evade me. We only did it once in my undergrad years and I haven't
touched plants at a cellular level since.

Sung, you got any protocols to share from P.patens?
PS Andreas, I like the use of Xylitol instead of mannitol! It's a plant
sugar, so shouldn't be very toxic, and it's bacteriostatic against lots
of common bacteria, so might help avoid excess contamination if it works
well..

On 22/11/12 20:49, Sung won Lim wrote:
> I think you induced too much stress in the cells. I don't see any
> protoplasts. Which plant are you working with, and do you know how large
> the protoplasts will be?
>
> -sung
> On Nov 22, 2012 1:16 PM, "Mega" <masterstorm123@gmail.com> wrote:
>
>> Hi,
>>
>> As the pectinase had finally arrived, I mixed some cellulase (around 10-20
>> uL) with pektinase (just some with a spatula)  in an 1.5 mL eppi. Then I
>> added 8% xylitol solution (isotonic, that the cells don't burst from
>> osmotic stress. I had no manitol like the instruction video said so I had
>> to use that).
>>
>>
>> Incubated for ca 90 minutes, and then microscoped some of it. Find the
>> pictures attached ;)
>>
>>
>> I think I will let the enzymes work over night with the rest, and then
>> centrifuge it, discard supernatant, add xylitol solution. And microscope
>> again.
>>
>>
>> Does anyone know if there are some protoplasts in it? Highest zoom = 400
>>
>> I belive to see there are some cells of which you see the chloroplasts.
>> But the ''chloroplasts'' might be as well be small entire plant cells in a
>> sugar coating?
>>
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