Sterilize bag of plates, media bottle, and scissors with 70% ethanol and place directly into the hood, then sterilize your gloves and keep your hands in the hood for the remainder of the protocol.
Stand up plates in the bag, with the tops down. cut off the bottom of the bag.
Flip the tower of plates, and slide the bag off the top. fold the bag up so nothing can get in the top and set aside in the hood.
Separate the plates in stacks of five. Open the media, and hold sideways so the cap isn’t straight up, to prevent dust falling in during plating.
Pick up all of the stack except the bottom half of the bottom plate, and pour media on until the plate is 2/3 covered. While continuing to hold the rest of the stack in hand, but the lid on that plate and remove the lid off of the new bottom plate, and pour again. Repeat until done.
Sulfuric Acid Scarification
1. Scarify seeds in small flasks with enough concentrated sulfuric acid to cover for 5-12 minutes
(we use the shorter time for sensitive mutants) — seeds are ready when small black spots are visible
2. Decant acid into a hazardous waste bottle
3. Rinse seeds with sterile water 4-5 times, putting first rinse in the hazardous waste bottle
4. Surface sterilize in concentrated Clorox + 1 drop Tween for 3 minutes
5. Add at least an equal volume of water
6. Decant liquid into sink with running water
7. Rinse with sterile water 4-5 times
8. Imbibe at room temp on an orbital shaker for 3-6 hours, changing sterile water several times
9. At this point seeds can plated for germination or can be stored in water at 4oC for 24-72 hours to improve germination, especially for recently harvested seed). Can also perform second sterilization with 10% bleach solution (make fresh) for 1 minute.
NOTES : Several rinses are needed to remove the Clorox, but it is possible to use a shorter time or even skip imbibing on the shaker, especially if seeds are to be put in the cold.
Modified from Sharon Long’s lab: http://cmgm.stanford.edu/biology/long/files/protocols/Germination.pdf
• Each section has a clear, descriptive heading of the experiments which were performed and detailed writing of the experimental observations, thoughts, and results.
• Each entry is dated.
• Each entry is legible.
• Each entry is in English.
• Each entry is written immediately after the work was performed.
• Multiple lab notebooks should be labeled numerically in the order of which they were written.
• Experimental data, originals or copies (e.g. micrographs), should pasted or taped into lab notebooks. If copies are made, originals should be organized in a separate binder on which the lab notebook number and page are denoted.
• On collaborations, clearly indicate who did what work and who was present for which experiments.
• At the end of a research project, all lab notebooks will be returned and archived in the group for use by as reference for future students.
The notebook itself is the property of Michigan State University. If you terminate your employment, you must turn this notebook over to your group.
See also: http://www.ruf.rice.edu/~bioslabs/tools/notebook/notebook.html, http://www.lanl.gov/orgs/tt/pdf/intell_property/nb_guidelines.pdf
TY (Tryptone Yeast)
Ingredient, g/L, g/600mL
tryptone, 6, 3.6
yeast extract, 3, 1.8
CaCl2, 0.38, 0.228
Dissolve first 3 ingredients in dH2O. If making solid media, add agar and **DO NOT MIX***—the autoclave will melt the agar for proper mixing.
bacto-agar, 16, 9.6
Autoclave on liquid 30min cycle (no exhaust). Remove promptly to a water bath: 50C will keep everything liquid; ~40C [hot bath] is the temperature you want to pour at.
use dH2O from the tap
dissolve before autoclaving to avoid crystals
for broth (liquid media), omit agar