Hello everyone! I am a student and I am trying to express fluorescently tagged proteins in Arabidopsis using the Tape-Arabidopsis Sandwich method. I isolated and got nice intact protoplasts. However the transfection is difficult to reproduce. Only in one out of 4 experiments I get transfected cells. Is there anyone that can suggest me what can be the crucial point for PEG mediated transfection or what I can do to get better results. I would really appreciate to learn from your experience.
We often use Tape-sandwich method to isolate protoplast and express tagged proteins and for protein-protein interaction. Our success rate with this method is more than 90%. We follow these tips
1. Health and age of the rosette: We only take 2-3 young leaves from a healthy rosette of 3-4 week. We avoid rosette in which flower bud is visible and too young and old leaves.
2. Size and concentration of the plasmid: If you need good transfection, use small plasmid. We use around 30 micrograms of good quality plasmid per transfection.
3. PEG: We use only this brand of PEG4000 (Fluka, #81240).
4. Handling: Be very gentle with protoplasts. During transfection, cut your pipette tip to reduce injury to the protoplast.
I hope these tips will be useful to you.
Good luck with your experiment!! :)
make sure the PEG solution is prepared at least 3 hours prior to transfection. This is one of the most important factors which is overlooked according to my experience. And be sure that the volume is accurate.
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