6. When working in the fume hood wear the appropriate personal
protective equipment of gloves and a long-sleeved lab coat.
Work with the bleach and acid separately. If either bleach or
acid splashes on gloves, change gloves before handling other
materials. Always remove gloves in the fume hood in case of
bleach or acid contamination.
7. The beaker should be at least twice the total liquid volume of
bleach + HCl. This prevents splashes escaping the beaker while
adding HCl, which can damage seeds, bleach clothes, or burn
exposed skin.
8. Monitor the container during sterilization to ensure chlorine
gas accumulation, which should be visible as a greenish-yellow
haze inside the container. Periodically check the container to
ensure that pressure inside has not unseated the lid or Parafilm
®
. If the lid has come unseated or the Parafilm
® has come
loose, close the lid and carefully wrap the container with an
additional layer of Parafilm
® .
9. Slowly add 1.5 g NaHCO 3 to the beaker containing the
bleach/HCl solution and stir with a glass rod to dissolve.
Continue adding NaHCO 3 until CO 2 gas bubbles have
stopped forming. Check if the pH of the solution is neutral,
and, if necessary, add additional NaHCO 3 until the pH is 7. At
this point the solution can be disposed of according to all
applicable disposal guidelines. If a smell is noticed during disposal, then the solution should immediately be returned to the
fume hood to begin the neutralization process again.
10. Thiamine and 2,4-D stock solutions must be added to the
media in a laminar flow hood to prevent contamination of
stock solutions. Preparing these two solutions have been
described previously [7].
11. Always work with cell cultures in a laminar flow hood. Mix the
culture well immediately before pipetting since the cells settle
to the bottom of the flask.
12. Development of cells into clumps will occur. A Magenta™ box
allows enough space for several tissue pieces to grow. Lack of
growth and change in color from healthy green to yellow can
be signs of an aging culture caused by having transferred old
cells. Keep 1 week old cell suspension culture and 2 weeks old
callus cultures at 4
C as backups if needed to restart cultures.
13. Growth of the callus after continued passage becomes independent of cytokinin [24].
14. For Arabidopsis or Brassicaceae similar in size: single plants can
be grown in 5.5 cm square pot or a flat insert (e.g., STI-804). A
98 well propagation tray (e.g., PROP-98-RD) can be used for
planting individual plants of many lines for genomic studies.
Handling Arabidopsis Plants
17
protective equipment of gloves and a long-sleeved lab coat.
Work with the bleach and acid separately. If either bleach or
acid splashes on gloves, change gloves before handling other
materials. Always remove gloves in the fume hood in case of
bleach or acid contamination.
7. The beaker should be at least twice the total liquid volume of
bleach + HCl. This prevents splashes escaping the beaker while
adding HCl, which can damage seeds, bleach clothes, or burn
exposed skin.
8. Monitor the container during sterilization to ensure chlorine
gas accumulation, which should be visible as a greenish-yellow
haze inside the container. Periodically check the container to
ensure that pressure inside has not unseated the lid or Parafilm
®
. If the lid has come unseated or the Parafilm
® has come
loose, close the lid and carefully wrap the container with an
additional layer of Parafilm
® .
9. Slowly add 1.5 g NaHCO 3 to the beaker containing the
bleach/HCl solution and stir with a glass rod to dissolve.
Continue adding NaHCO 3 until CO 2 gas bubbles have
stopped forming. Check if the pH of the solution is neutral,
and, if necessary, add additional NaHCO 3 until the pH is 7. At
this point the solution can be disposed of according to all
applicable disposal guidelines. If a smell is noticed during disposal, then the solution should immediately be returned to the
fume hood to begin the neutralization process again.
10. Thiamine and 2,4-D stock solutions must be added to the
media in a laminar flow hood to prevent contamination of
stock solutions. Preparing these two solutions have been
described previously [7].
11. Always work with cell cultures in a laminar flow hood. Mix the
culture well immediately before pipetting since the cells settle
to the bottom of the flask.
12. Development of cells into clumps will occur. A Magenta™ box
allows enough space for several tissue pieces to grow. Lack of
growth and change in color from healthy green to yellow can
be signs of an aging culture caused by having transferred old
cells. Keep 1 week old cell suspension culture and 2 weeks old
callus cultures at 4
C as backups if needed to restart cultures.
13. Growth of the callus after continued passage becomes independent of cytokinin [24].
14. For Arabidopsis or Brassicaceae similar in size: single plants can
be grown in 5.5 cm square pot or a flat insert (e.g., STI-804). A
98 well propagation tray (e.g., PROP-98-RD) can be used for
planting individual plants of many lines for genomic studies.
Handling Arabidopsis Plants
17