2. EDTA can only be dissolved at a basic pH. Add NaOH pellets
and wait for a few minutes. Then add additional pellets one by
one until the EDTA powder is dissolved.
3. Boric acid precipitates after long storage (around 1 month).
Thus, preparing a large volume of 10Â TBE is not advised.
4. Heating is required to dissolve SDS.
5. SDS can precipitate if the room temperature decreases below
20
C. Heat the solution to dissolve SDS before use, if needed.
6. Pipetting with normal tips exposes protoplasts to an excessive
osmotic pressure change and can smash them. Better use pipettes with wide-mouth tips, and cut micropipette tips with
scissors to enlarge the opening hole. Avoid pipetting and rather
pour protoplast solutions to the new containers if possible.
7. Applying braking combined with centrifuge force can smash
the cells.
8. Add the buffer slowly along the wall of the tube or the Petri
dish to avoid excess of pressure and mechanical force, which
could smash the protoplasts.
9. Quality of the seeds is important. Better to use freshly harvested seeds (less than 1 year old). If you use old seeds, sterilization is less efficient. In the case of old seeds or mutant seeds,
we have used 80% ethanol and 4% Tween™ 20 (rather than
70% ethanol, 0.1% Tween™ 20) as sterilization solution. Please
note that times must be respected to avoid damage to the seeds.
10. Intact protoplasts are perfectly round and well isolated (see
Fig. 1). Their size can vary, but both large and small ones can
be counted if they are not damaged. The number of protoplasts
prepared from 10 mL of cell culture is usually around 2 Â 10
6
whereas 200 mg of seedlings will give about 1 Â 10
6
protoplasts.
11. The transfection buffer must be freshly prepared on the day of
transfection.
12. Protoplasts can be kept in the 12-well plate for a maximum of
30 h. The longer the protoplasts are kept in the plate, the more
of them will die.
13. LiCl causes the precipitation of large RNAs (i.e., >200 nt in
size). After centrifugation, the supernatant contains tRNAs
and small noncoding RNAs (including tRFs).
14. One hour is usually sufficient for precipitation if ethanol kept at
À20
C is used. Otherwise, precipitation can also be performed
overnight.
15. To avoid bubbles, the membrane should be wet before placing
the gel on it. Bubbles can be chased by smoothly pressing a
pipette on the gel.
428
Ste ´ phanie Lalande et al.
and wait for a few minutes. Then add additional pellets one by
one until the EDTA powder is dissolved.
3. Boric acid precipitates after long storage (around 1 month).
Thus, preparing a large volume of 10Â TBE is not advised.
4. Heating is required to dissolve SDS.
5. SDS can precipitate if the room temperature decreases below
20
C. Heat the solution to dissolve SDS before use, if needed.
6. Pipetting with normal tips exposes protoplasts to an excessive
osmotic pressure change and can smash them. Better use pipettes with wide-mouth tips, and cut micropipette tips with
scissors to enlarge the opening hole. Avoid pipetting and rather
pour protoplast solutions to the new containers if possible.
7. Applying braking combined with centrifuge force can smash
the cells.
8. Add the buffer slowly along the wall of the tube or the Petri
dish to avoid excess of pressure and mechanical force, which
could smash the protoplasts.
9. Quality of the seeds is important. Better to use freshly harvested seeds (less than 1 year old). If you use old seeds, sterilization is less efficient. In the case of old seeds or mutant seeds,
we have used 80% ethanol and 4% Tween™ 20 (rather than
70% ethanol, 0.1% Tween™ 20) as sterilization solution. Please
note that times must be respected to avoid damage to the seeds.
10. Intact protoplasts are perfectly round and well isolated (see
Fig. 1). Their size can vary, but both large and small ones can
be counted if they are not damaged. The number of protoplasts
prepared from 10 mL of cell culture is usually around 2 Â 10
6
whereas 200 mg of seedlings will give about 1 Â 10
6
protoplasts.
11. The transfection buffer must be freshly prepared on the day of
transfection.
12. Protoplasts can be kept in the 12-well plate for a maximum of
30 h. The longer the protoplasts are kept in the plate, the more
of them will die.
13. LiCl causes the precipitation of large RNAs (i.e., >200 nt in
size). After centrifugation, the supernatant contains tRNAs
and small noncoding RNAs (including tRFs).
14. One hour is usually sufficient for precipitation if ethanol kept at
À20
C is used. Otherwise, precipitation can also be performed
overnight.
15. To avoid bubbles, the membrane should be wet before placing
the gel on it. Bubbles can be chased by smoothly pressing a
pipette on the gel.
428
Ste ´ phanie Lalande et al.