They formed clumps, but were they broken? Have you checked them under microscope to see whether they were still intact?

Did you adde DNA alone or with PEG?

Prof. Yuan-Yeu,

The protoplasts were broken. After PEG transformation I had some protoplasts in MaMg solution remaining. I kept that remaining protoplast in 4C and 24 hrs later I checked that protoplast also.  Interestingly, they were perfect, only DNA & PEG added protoplasts were broken.

Dr. Jafargholi Imani,

I added PEG solution. I made fresh PEG solution ( 400mM Mannitol, 100 mM CaCl2 and 40% PEG 4000) and mixed for 2 hours using a twist shaker.

I found somewhere that PEG solution should be mixed for atleast 2 hours, because it takes long time to stabilize the pH.

In this case, the clumping is  normal. You have to pellet the PP (by centrifugation) and after that remove the supernatant that contains PEG. The pellet should be then resuspended in PP medium.

Good luck

Dr. Jafargholi Imani,

I followed following protocol after adding MaMg solution. Still protoplasts were clumped up and ruptured.

• Add 300 µl MaMg Solution for each sample (Normally, two plates WT plants contain the protoplast equals to 5 samples, so add 2 ml MaMg solution) and suspend with rotating the tubes several times at an almost-horizontal position by hand.
• Take a 13 ml tube, add 30 µg Vectors containing gene of interest, then add 300 µl protoplast in MaMg solution in each tube, mix gently and completely by rotating the tubes several times at an almost-horizontal position by hand. This step is very important because it will prevent protoplasts from being coagulated during the next two steps.

• Add 300 µl PEG 8000 solution. Mix gently and completely by rotating the tubes at an almost-horizontal position by hand. Incubate at room temperature (22°C would be best, do this under Air Cooler) for 30 minutes.

• Add 1 ml W5 solution, mix by hand-revolving and incubate from 10 minutes.

• Add 1.5 ml W5 solution, mix by hand-revolving and incubate from 10 minutes.

•  Add 2 ml W5 solution, mix by hand-revolving and incubate from 30 minutes.

•  Centrifuge at 530 rpm for 4 minutes and resuspend with 2 ml W5 solution.

• Incubate at 23°C for 20-24 Hours (Using the Tissue Culture Room).

I wonder your DNA purity. DNA purity is very important for the protoplasts transformation mediated with PEG. I recommend using DNA maxi or midi prep kit for DNA purification. It will improve your work. 

I agree with Young Jun. I had a lot of problem with DNA purity. 

Residual salt in preped DNA may cause clumping. I used MN midi kit and Qiagen midi.

Both worked, but both occasionally caused clumping.  

Thanks a lot Dr. Young Jun and Dr. Young-Min

I was wondering about Residual salt. I have used Qiagen Midi Prep Kit. Although My vectors are high copy, but I didn't get good concentration. So, I did one more time EtOH precipitation. During EtOH precipitation I have used 3M sodium acetate litttle bit higher concentration than usual (200 micro liter 3M NaOAc for 1 ml EtOH).

Is there any chance that some residual salt remains in the DNA?

After precipitation, I washed my pallets twice with 70% EtOH. So, the salt should be removed.

I don't like EtOH ppt for protoplast transfection.

Similarly, I also couldn't get enough DNA concentration because it was binary vector.

So I decided to do subcloning into cloning vector....to avoid ppt...

@  A K M Mahmudul Huque,

Suggestion #1:

Since you went through 'concentrating' procedure (using 3M NaOAc), you might have some residual salt remains in the DNA sample. This may not be good for your protoplasts. When I concentrated my plasmid DNA, I used a 'Vacuum' machine (a.k.a. "SpeedVac") to remove water (in cold). Besides, my plasmids were eluted in water, not in TE (to avoid any salt). I aliquot my plasmid DNA in several tubes for storage, to reduce freeze-and-thaw cycles, which degrades DNA.

See attached for a SpeedVacTM Concentrator machine. Nowadays, there is a newer and smaller model.

Suggestion #2:

When we do protoplast transformation, we co-transfer some 'carrier' DNA (such as salmon sperm DNA) with our plasmids-of-interest to protect plasmids from being degraded once them get into protoplasts. In the meantime, we always include one or two 'negative controls', where the protoplasts are transformed with carrier DNA only (without plasmid-of-interest). If you are worried about your plasmid samples' purity (such as salt residue in them), you should include these controls in your experiments. Ready-to-use commercial carrier DNA can be used for this purpose. Use same amount of DNA for each to do transformation side by side. If no broken protoplasts in the controls, but observe broken protoplasts in the "plasmid" experiment, then your plasmid sample may have problem. If both failed (busted protoplasts), then probably there are other causes which leads to broken protoplasts. Theoretically, the commercial DNA should not give you troubles.

@ Prof. Yuan-Yeu,

Thank you very much your suggestions. I believe it would be really helpful. Especially, commercial DNA as a control. I would try them. Hope this time I can get some good data.

Dr Yuan ,

I am also observing a similar problem , But the difference is that clumps suddenly start to form after 12 hours or more,. We usually get 60 -80 % transfection efficiencies deduced on the basis of GFP signals. After transformation the protoplasts appear to be normal. We have been using the Sheen J protocol. But the only difference is we are using brassica napus protoplasts. Our lab also routinely works with arabidopsis protoplasts but similar problems are also observed there some times.

Thanking in anticipation.

@ Muhammad Saad Rehmani,

Thank you for sharing your experience with us. Did the aggregation of your brassica napus protoplasts affact your downstream experiments (or your project)?

Yes it does affect my project because i need to perform different viability assays such as FDA and EVANS using a fluorescence microscope. and you cannot take pictures of big clumped dead cells. i thought it maybe due to the GOI at first. But GUS samples also showed clumping. For other experiments such as protein detection it doesn't pose a problem and other fellows surely have detected proteins. Sorry for replying late , there are browsing issues over here and i couldn't post a reply.