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Protoplast Isolation

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Protoplast Isolation

Postby ev350 » Thu Sep 08, 2011 8:23 pm

Hello there forum.
I am new here and studying under the HL IB programme. I have chosen to do an extended essay in biology, obviously, and I am requesting some assistance on the topic. My experiment involves the isolation of protoplasts from lettuce leaf. The second part of the experiment is the fusion of the protoplasts, but I won't get into that as my problem comes before this.

First off, it will be hard to explain what exactly could go wrong and that is where I am asking for help. Therefore, I will try to explain my procedure as accurately as I can and label which equipment I am using:

Solutions:
13% Sorbitol Solution - This is made by adding 13 grams of sorbitol powder to 100cm3 of water, is it not?
21% Sucrose Solution - This is made by adding 21 grams of sucrose puriss. powder to water, is it not?
Carbohydrase enzyme mixture - is this just Viscozyme?


1. Cut a piece of lettuce leaf into roughly 5mm x 5mm using scissors.
2. Add 15-20 lettuce pieces to 9.5cm3 13% sorbitol solution in a test tube.
3. Incubate the test tube in a water bath, set at 37C, for 5 minutes. <-- How accurate does the temperature have to be?
4. Gently stir 0.5cm3 of the carbohydrase enzyme mixture into the test tube.
5. Return the tube to the water bath for another 20 minutes. Gently agitate the tube from time to time during incubation. <-- Is the gently stressed? Should it be very gently?
6. Tightly pack the spout of the filter funnel with a nylon gauze. <-- would a pair of tights suffice?
7. Pour the contents of the test tube into the filter funnel.
8. Wash any trapped protoplasts through the filter with 10cm3 of 13% sorbitol solution. Collect all the filtrate in a centrifuge tube.
9. Balance the centrifuge tubes then spin for roughly 5 minutes at 2,000 rpm. <-- does it have to be exactly 2,000 rpm? My school provides with me an MSE minor centrifuge from the late 50's. It has numbers from 1 to 10. Therefore I spin at around 7-8 and pray it works. I called the company and said that the unit is a bit 'hit and miss' considering its age and create period. They said it could reach 4,000 rpm, but were unable to clarify.
10. Carefully discard the supernatant, leaving the pellet of protoplasts at the bottom of the tube. <-- This is where my error occurs. I do not receive a pellet, there is nothing on the bottom of my centrifuge tube.
11. Resuspend the pellet in approximately 0.1 cm3 of 21% sucrose solution.

After completing all this, when looking through the microscope, it appears that there are perhaps, but i am unsure, a few protoplasts, maybe 3-4. Although, they do seem burst. There what appears to be quite a bit of debris on occasion as well. <-- This is the 6th time performing the experiment.

I hope this is enough information for you to try and make an educated guess of where I could be going wrong.

Thank you for your time,
ev350
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Postby canalon » Fri Sep 09, 2011 1:13 pm

Enzymes: I do not know
3- However precise you can be. Within 1° or 2° is probably OK
4- you want your stuff being homogeneous as they will settle, but be gentle as you are dealing with membranes that are not used to be without cell walls or in a very carefully controlled environment. So as gentle as you can while still achieving the mixing effect. So not a vortex at full power
5- Probably yes. As long as you do not use the fishnet kind ;) But you want the simple kind, it is to filter out the solids (undigested bit of leaves) with minimum retention. So not the winter kind with mixed fabric.
6- I see that you have the same problem I had in another lab. Similar kind of old stuff. There are 2 problems with those: no way to accurately measure the speed and they do not refrigerate. I would suggest that in your case, you should go for the experimental method: run 5 minutes at 5. If you seee nothing,5 more minutes at 6, etc. You might have to go to 10 because the speed you have might be for a larger rotor (ie the g forces might be stronger) or you can try to increase centrifugation time. And if you have access to a cold room or a fridge in which you can put and run the centrifuge use that rather than room temperature.
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)
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Postby JackBean » Mon Nov 21, 2011 11:21 am

9 use rather slower speed (since you do not want to break your protoplasts), but for longer period, that should give the protoplasts enough time to settle down and form pellet.
11. since they seem to burst, you have probably too low ionic strength in your solution. Better use slightly higher ionic strength, that should assure that your protoplasts won't burst.
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