Griffitts:Conetainers: Difference between revisions
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** Make sure that all the roots from previous experiments have been removed—this may require scrubbing each one with the brush | ** Make sure that all the roots from previous experiments have been removed—this may require scrubbing each one with the brush | ||
* When they are all clean add bleach until you can smell it easily (about 1.5 L) | * When they are all clean add bleach until you can smell it easily (about 1.5 L) | ||
* Allow them to soak for | * Allow them to soak for several hours | ||
** We used to do this overnight, but the bleach was corroding the sinks | |||
* After they are done soaking, take them out, and place them on a cart covered with benchcoat and leave them in the autoclave room for an additional day to dry | * After they are done soaking, take them out, and place them on a cart covered with benchcoat and leave them in the autoclave room for an additional day to dry | ||
* Wipe down the rack well with 70% ethanol | * Wipe down the rack well with 70% ethanol | ||
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* On the second day of [[Griffitts:Conetainers#Watering|watering]], remove Saran wrap and only use the dome from then on to cover them | * On the second day of [[Griffitts:Conetainers#Watering|watering]], remove Saran wrap and only use the dome from then on to cover them | ||
<div class="noprint"><gallery> | <div class="noprint"><gallery> | ||
Image:Hydrated Seedlings.JPG|Add water to germinated seedlings to keep them from drying out while planting. | |||
Image:Griffitts-Hydrating turface.JPG|Wet the turface before planting. | Image:Griffitts-Hydrating turface.JPG|Wet the turface before planting. | ||
Image:Griffitts-Digging in turface.JPG|Use sterile tweezers to dig a hole in the wet turface. | Image:Griffitts-Digging in turface.JPG|Use sterile tweezers to dig a hole in the wet turface. | ||
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** During the third week, water '''every other day''' with 1X [[Griffitts:Common media#Broth|SNM]] | ** During the third week, water '''every other day''' with 1X [[Griffitts:Common media#Broth|SNM]] | ||
** During the last week, water '''every day''' (except the day before you want to record your results) with dH<sub>2</sub>0 | ** During the last week, water '''every day''' (except the day before you want to record your results) with dH<sub>2</sub>0 | ||
<div class="noprint">[[Image:Griffitts-Marked 25 mL pipette.JPG|center]]</div> | |||
==Inoculation== | ==Inoculation== |
Latest revision as of 09:41, 5 April 2010
Turface preparation
Note: For Best results do turface one week prior to desired planting day so that it has time to dry
- Get two large white tubs and fill them ½ full with Pro League turface
- Rinse the turface repeatedly until the water comes off clear
- Your goal is to remove the smaller particles as well as leaves, roots, and salt buildup left over from prior experiments
- Drain off all of the water and autoclave
- For small loads, use the autoclave in 758 WIDB
- 30 minutes sterilize time, 30 minutes dry time
- For large loads, use the autoclaves in 740 WIDB
- 60 minutes sterilize time, 99 minutes dry time
- If possible leave in the autoclave overnight for additional drying
- For small loads, use the autoclave in 758 WIDB
- Remove from autoclave and immediately cover with foil to prevent contamination
- It will be hot, so bring orange autoclave gloves
Cone preparation
- Fill the sink up with water and place a rack with 200 cones into it upside down
- Make sure that all the roots from previous experiments have been removed—this may require scrubbing each one with the brush
- When they are all clean add bleach until you can smell it easily (about 1.5 L)
- Allow them to soak for several hours
- We used to do this overnight, but the bleach was corroding the sinks
- After they are done soaking, take them out, and place them on a cart covered with benchcoat and leave them in the autoclave room for an additional day to dry
- Wipe down the rack well with 70% ethanol
- Wrap white tape around the top of the rack so you can label each row
- Wipe down the cover with 70% ethanol
- Proceed to batting square preparation
Batting square preparation
NOTE: This should be done the same day as the cone preparation
- Cut the white batting into 200 squares (~1 square inch each) for each conetainer being prepared
- They sometimes stick together, so it doesn't hurt to cut a few extra
- Soak them overnight in a small tub with 4 L dH2O and 130 mL bleach
- Cover with aluminum foil to prevent contamination
- While wearing gloves, remove the squares from the bleach and squeeze them dry
- Place them on a cart covered with benchcoat and leave them in the autoclave room for an additional day to dry
- This can be the same cart that the conetainer rack is drying on
- When the squares are dry, stuff them into the bottom of the cones using sterile technique (a sterile inoculating stick is good for this)
Rack preparation
- Wipe down a funnel and a scoop with 70% ethanol
- Using the sterile funnel and scoop, fill each cone to the top with turface
- Compact the turface by lifting it and pounding it against the counter
- Fill each cone again until it is about 1 cm from the top of the cone
- It won’t compact much the second time, so be sure not to overfill
- Put on gloves and spray them with 70% ethanol
- Wipe off any extra turface not in the cones
- Cover rack with Saran wrap until you are ready to plant and place dome on it
Planting
- Add 3 mL sterile dH2O to a row of ten cones
- Using flamed tweezers, dig a hole in the center of each of the cones
- Remove parafilm from the germinated plants and add enough dH2O to the plates to allow all the plants to be kept moist
- Using the flamed tweezers, insert the appropriate plant into the desired cone
- It may sometimes be desirable to plant two plants per cone
- Be sure not to force the plant into the turface, so that you don’t damage the root
- Using the flamed tweezers, compact the turface around the plant
- You want the top of the cotyledon showing and that is all
- After planting all 10 cones, add another 3 mL sterile dH2O to each cone
- Proceed to the next row of cones
- After you have planted the entire rack, add another 3 mL sterile dH2O to each cone
- Cover with Saran wrap, top with the dome, and place on the light bank
- On the second day of watering, remove Saran wrap and only use the dome from then on to cover them
Watering
Note: On average we set an experiment for 25–28 days
- Use a black marker to mark ten 3-mL intervals on a 25-mL pipette
- Being careful not to touch the rack, turface, or plants with the pipette, dispense 3 mL of water or SNM per cone
- Do not reuse 25-mL pipettes
- Watering schedule:
Inoculation
Note: Inoculation day is four days after planting, so you will want to make sure that you have steps 1 and 2 done by Day 4 of the experiment
- Grow up all your bacterial strains to singles
- This takes 2–3 days
- From those singles patch out an ~1 cm square patch
- Fill a Falcon tube with 1 mL more of dH2O than the number of plants you will be inoculating with the same bacterial strain
- Put enough bacteria into the tube to make the solution slightly opaque
- Vortex
- Add 1 mL of the bacteria solution to each plant
- Be sure to dig the pipetter ~0.5 cm into the turface so that it doesn’t splash
- Make sure you keep track of where you are so you don’t inoculate the wrong plants!
- Repeat Steps 3–6 for all bacterial strains