These methods have been developed by the Phytomorph lab at Doane University to provide researchers with more convenient means to analyze plant genome functions.
Video footage and photos of all methods developed in the lab were recorded and edited into easy to follow tutorials.
Minimal Media Preparation * Agar & Plate Preparation * Seed Preparation & Storage * Label Production * Seed Planting * Seed Stratification & Growth * Equipment * High-throughput Imaging of Root Gravitropism
Minimal Media Preparation
- 1000 µl pipette
- 1 1000 mL beaker
- 2 1000 mL graduated cylinders
- 100 X MES (methyl-ethyl-sulfate)
- pipette tips
- 1000X KCl and CaCl2
- RO Water (Reverse Osmosis)
- 2 Spin Bars
- pH Indicator
- Kimwipes
- BTP Buffer (bis-tris propane)
- 2 storage bottles
1) Preparing 2L Minimal Media (1mM KCl, 1mM CaCl2, 5mM MES, 1% agar, pH=5.7 with BTP)
| 1.1) | Obtain a 1000 µL pipette, and one 1000 mL beakers. |
| 1.2) | Set the pipette to 1000 µL. |
| 1.3) | Add 20 mL 100x MES ((0.5M) methyl-ethyl-sulfate), solution to the 1000 mL beaker. |
| 1.4) | Dispose of tip in biohazard bag, place new tip on pipette, and add 2000 µL of 1000x KCl and CaCl2 solution to the same beaker. Dispose of tip in biohazard bag. |
| 1.5) | Fill the beaker containing solution with RO water until it reaches the 800 mL mark. |
| 1.6) | Sterilize a spin bar with 70 % EtOH solution and place in the beaker containing solution. |
| 1.7) | Place the beaker on a stirring plate and set the dial to 7. |
| 1.8) | Check the pH of the solution with a pH indicator by removing the cover of the indicator, gently wiping with a Kimwipe®, and placing in solution. |
| 1.9) | Turn up the stir dial until the solution moves in a circular motion. |
| 1.10) | To alter the pH of the solution, pipette BTP (bis-tris propane) buffer dropwise into the solution until pH reaches exactly 5.70. This should be done very slowly, as pH changes drastically with each drop. |
| 1.11) | When pH reaches 5.70, remove indicator from solution, rinse with RO water, wipe with a Kimwipe®, and place in cleaning solution. |
| 1.12) | Fix the spin bar to the bottom of the beaker by holding a larger bar on the outside of the beaker. |
| 1.13) | Pour the solution into 1 1000 mL graduated cylinder. |
| 1.14) | Rinse the beaker that contained the solution with RO water, and pour in the graduated cylinders. (Do this at least 3 times) |
| 1.15) | Fill the cylinders with RO water to the 1000 mL mark. |
| 1.16) | Mix the solution by placing a sterilized spin bar in the graduated cylinder, then placing on a stir plate. When mixed, pour 500 mL of the solution into another 1000 mL graduated cylinder. |
| 1.17) | Bring the volumes of both solutions to 1000 mL with RO water and mix as in 1.16. |
| 1.18) | Pour solutions in labeled bottles, and set aside for future use. |
Agar and Plate Preparation
- 1000 mL Minimal Media (1 mm KCl, 1mm CaCl2, 5mm MES (methyl-ethyl-sulfate)
- 2000 mL Erlenmeyer flask
- 1 graduated cylinder
- Difco agar, granulated
- 1 Weighing boat
- Autoclave and Tinfoil
- ≈130 petri dishes
- Hot plate
- 60 mL syringe
- stop-cock attachment
- 70 % EtOH
- Large storage containers
2) Agar and Plate Preparation
| 2.1) | Measure out 1000 mL of Minimal Media (1 mM KCl, 1mM CaCl2, 5mM MES (methyl-ethyl-sulfate), pH 5.7 with BTP (bis-tris propane), 1% agar) in a graduated cylinder then pour into a 2000 ml Erlenmeyer flask. Measure 300 more ml of media in the graduated cylinder. |
| 2.2) | Weigh out 13 g of the Difco Agar, Granulated and add to the Erlenmeyer flask. DO NOT weigh extra. |
| 2.3) | Use the remaining 300 ml of the Minimal Media to get the last of the granulated agar from the weigh boat. Swirl the solution in the Erlenmeyer flask so that it dissolves as much as possible. |
| 2.4) | Put tin foil on the top of flask and place it in the Autoclave. |
| 2.5) | Set the Autoclave to the liquid #2 setting with the temperature at 121°C and time at 12-15 minutes. |
| 2.6) | While the agar solution is in the autoclave, set up approximately 130 Petri dishes to pour the agar into when it is done in the autoclave. |
| 2.7) | After the solution is done autoclaving, place it on a heating plate set at 89°C to prevent solidification. |
| 2.8) | Rinse a 60 mL syringe and stop-cock valve with a 70% ethanol solution for sterilization prior to plate preparation. |
| 2.9) | Pour the agar solution into the sterilized syringe (total 60 mL) with stop-cock attachment. Leave the syringe plunger out. |
| 2.10) | Pour 10 mL of agar into each petri dish. The 60 mL syringe will only allow the pouring of 6 dishes. |
| 2.11) | After 6 plates are poured, swirl the plate filled agar so that it completely covers the bottom, and place the lid on top. Hold the plate a few inches off the preparation surface to prevent scratching of the dish. |
| 2.12) | Repeat steps 2.9-2.11 until the agar runs out. |
| 2.13) | After all of the plates are poured, swirled, and covered let them sit for a couple hours so the agar can solidify. |
| 2.14) | Place petri dishes in containers to prevent the agar from drying out. |
Label Production and Tools
- Barcode font
- Microsoft Office word 2007
- Microsoft Office Access 2007
- Avery White File Folder Labels 5366
- Printer
- Previously poured agar plates
- Label Template
- Large storage containers
Get a complete listing of tools
4) Label Production
| 4.1) | If one would like to have the barcode font as a part of the labels, proceed with step 2.2, if not, skip to step 2.4. |
| 4.2) | Download a barcode font such as Code 128 from an appropriate source. |
| 4.3) | Follow the company instructions for installation of the program into Microsoft Word. |
| 4.4) | Open up Microsoft Word (These directions refer to Microsoft Word 2007). |
| 4.5) | Under the Mailings tab click on the Start Mail Merge button under the Start Mail Merge submenu then choose Labels. |
| 4.6) | Choose the appropriate label vendor and product number indicated on label package. Click OK. |
| 4.7) | Click on Select Recipients from the Start Mail Merge submenu. |
| 4.8) | Choose Use Existing List and locate the database containing the metadata associated with one's experiment. Note: Database information and fields are based upon the user's discretion appropriate for the experiment. |
| 4.9) | From that database, choose the sheet containing the specific information that should be incorporated on the labels. e.g. Plate Properties. |
| 4.10) | Insert fields from the chosen sheet in order to lay out the desired label format. |
| 4.11) | To insert a field, click on Insert Merge Field from the Write & Insert Fields submenu. Choose the desired field/fields in dropdown box. To preview results choose the Preview Results button in the Preview Results submenu. Alignment, font, size etc. can easily be adjusted in this view. e.g. barcode font |
| 4.12) | After the first label is formatted as desired, click on Update Labels under the Write & Insert Fields submenu. |
| 4.13) | To preview the labels, click the desired record number in the Go To Record input box under the Preview Results submenu. Note: the record number is not the same as the unique ID number in the Access Database. The record number is in the lower left hand corner of the Access sheet. |
| 4.14) | To print the labels, click on Finish & Merge in the Finish submenu. Choose Print Documents and type appropriate record number range in the dialogue boxes. Click OK. |
| 4.15) | Set the desired printer settings. Click OK. |
| 4.16) | Place labels on previously made agar plates using designated template. |
| 4.17) | Organize labeled agar plates in storage containers to prevent drying out and for planting convenience. |
Seed Planting and Tools
- 3 pieces of filter paper
- Previously made agar plates with labels
- Prepared seed stocks (RIL or NIL)
- 100 µl pipette
- Pipette tips
- Seed sterilizing solution (70 % EtOH, 1% Triton X)
- 70% EtOH
- Planting template
- Planting tool
- RO water (Reverse Osmosis)
- Black permanent marker
- Microcentrifuge tubes
- Rubberbands
Get a complete listing of tools
5) Planting Seeds
| 5.1) | Place 3 pieces of filter paper on workspace, check plate label for appropriate seed numbers, and mark each filter paper with the appropriate seed number. |
| 5.2) | Find those seed line numbers on a printed list of seed numbers, and determine how many of each seed are needed. Note: For this experiment, three seeds are used for each designated seed line. e.g. if there are three number 34's on the printed list, 9 seeds from tube 34 are needed. |
| 5.3) | Pour the correct amount of seed on the coinciding filter paper |
| 5.4) | Using a 100 microliter pipette, saturate the seeds with seed sterilizer (70% EtOH, 1% Triton X), then, using a different tip, cover them with 70% ethanol. |
| 5.5) | Line up the labeled agar plate on designated template. |
| 5.6) | Dip planting tool in microcentrifuge tube filled with water, gently pick up specified seed, and place on the surface of the agar. Note: be very careful with this step as we don't want to damage the agar in any way. |
| 5.7) | Make sure to line the seeds up exactly with the pen dots on the template. It's also very important to pay attention to the seed numbers on the label and the order you're placing them on the surface of the agar. The seed lines should match up with those on the label. |
| 5.8) | After all 9 seeds are planted, make a vertical black line on the outside of the agar plate (use the template as a guide) using permanent marker. |
| 5.9) | Place the lid on the agar plate and secure the bottom of the petri dish by wrapping parafilm around the edge of the plate. |
| 5.10) | If any extra seeds were sterilized for future plates, place them in separate designated microcentrifuge tubes. e.g. If the filter paper labeled 34 has 6 extra seeds, place them in a separate tube for sterilized seeds labeled 34 small or large. If there is no tube with that number, make a new one. Pay attention to whether you're working with small seeds or large seeds. |
| 5.11) | Repeat steps 5.1-5.10 with each new agar plate and label. |
| 5.12) | When all plates for that day have been planted, stack them numerically in groups of 6. |
| 5.13) | Place the plates in the refrigerator on the day designated by the label, and let stratify for 5 days. |
Seed Preparation, Storage, and Tools
- Designated seed stock (RIL or NIL)
- Microcentrifuge tubes (various colors)
- Seed Scoop
- Sieves (multiple sizes)
- Motorized sieve shaker
- Sieve brushes
- Wood teasing needle
- 70% EtOH
- Seed Storage Device (Storage containers with silica gel filled nylons)
Get a complete listing of tools
3) Seed Preparation and Storage
| 3.1) | Combine equal amounts of seed from each NIL tube line into a clean centrifuge tube using designated scoop. |
| 3.2) | Determine the seed size needed for imaging, and select certain sieves for accurate seed separation. e.g. 212 micron and 316 micron 3" sieves were used to separate large and small seeds in this project. The seeds smaller than designated size were thrown out. |
| 3.3) | Wipe down designated sieves with 70% EtOH to prevent static electricity. |
| 3.4) | Sieve seed stocks. |
| 3.5) | Place small and large seeds in centrifuge tubes that are easily distinguishable from each other. Be sure to label tubes with designated seed line. |
| 3.6) | Store seed stocks in Tupperware with silica gel filled nylons to prevent seeds from drying out. Place in refrigerator. |
Seed Stratification and Growth
Transferring Plates to Growth Chamber
- Notebook
- Mechanisms
---- Square petri dishes
---- Small round petri dishes
---- Rubberbands
6) Moving Plates from Refrigerator to Growth Chamber
| 6.1) | Remove agar plates containing planted seeds from refrigerator after stratification period. |
| 6.2) | Record all plate numbers removed from refrigerator in log book |
| 6.3) | To prepare plates for entry into the growth chamber, place in mechanisms. |
| 6.4) | Mechanisms are constructed by placing one half of a square Petri dish face down on counter then placing planted agar plate on top. Next, place half of a small round Petri dish on top of the plated agar plate, then place another planted agar dish on top, another small round Petri dish, and a third planted agar plate. Finally, place the other half of the square Petri dish face up on top of the third planted agar plate. Note: Make sure all labels and seed lines are aligned. Secure mechanism with two rubber bands also in line with labels and seeds. |
| 6.5) | Record plate numbers in growth chamber log book. |
| 6.6) | Place mechanisms in growth chamber following designated organization pattern. Let grow for designated time period. |
Equipment
| Tools | Suggested Vendor/Website | Approximate Price (USD) | Notes |
| Epson Perfection V700 Photo Scanners | Epson | $600.00 | |
| Plexiglas Scanner Template | Handmade (see attached schematic) | $100.00 | (Attachment to Scanner Template File) - Tools Folder |
| Smart Strap Bungee Cords | Walmart
|
$10.48/10 pk | |
| Brinks Digital Outdoor Timers #42-1014-2 | Walmart | $10.00 each | |
| Duck Non-adhesive shelf liner #1369238 | Walmart | Price Varies | Placed under storage racks to prevent plates from slipping. |
| Storage Racks | Handmade | $25.00 | These were made by gluing two cookie racks together. We ordered 2 of the Norpro 19x13 Deluxe Cooling Racks |
| Supplies | |||
| 3M Micropore Tape #19-061-655 | Fisher Scientific | $0.49 each | |
| Triton X100 | Sigma-Aldrich X100-100ML | $30.50 |
| Tools | Suggested Vendor/Website | Approximate Price (USD) | Notes |
| Seed Scoop | Handmade - How-to | Instructions- Handmade Tools Protocols | |
| Motorized Sieve Shaker for 3" Sieves H-4326 | Humboldt Mfg | $230.00 | |
| 3" Sieves | Humboldt Mfg | $29.40 | |
| 3" Sieve Cover H-3913BC | Humboldt Mfg | $11.20 | |
| 1" Bottom Pan H-3913P | Humboldt Mfg | $13.30 | |
| Sieve Brush H-3774 | Humboldt Mfg | $13.50 | |
| Custom 3" Sieves | Advantech Mfg | $97.75 | |
| Teasing Needle | Any Biological Supply Co. | $0.20 | |
| Supplies | |||
| Whatman Filter Paper (110 mm) 09-851C Whatman #1541-110 |
Fisher Scientific | $44.59/100 | |
| 1.5㎕ Microcentrifuge tubes #1615-5599 | USA Scientific | $12.95 | |
| Compact PCR Tube Rack #2300-9602 | USA Scientific | $29.25 | |
| Avery White File Folder Labels 5366 | Any Office Supply Store | $45.00/1500 | |
| Silica Gel AC35740-0010 Acros Organics #357400010 |
Fisher Scientific | $58.81 | |
| Panty Hose | Any Department Store | $5.99-10.00 | |
| Storage Containers for Seed Storage | Any Supply Store | $5.00-15.00 |
| Tools | Suggested Vendor/Website | Approximate Price (USD) | Notes |
| HP Deskjet 6540 Color Inkjet Printer | amazon.com | apprx. $ 65.00 | |
| Label Template | Handmade - How-to | Picture and instructions on how to make -Handmade Tools Protocols File | |
| Supplies | |||
| Avery White File Folder Labels 5366 | Any Office Supply Store | $45.00/1500 | |
| Plastic Containers for Plate Storage | Any Supply Store | $10.00-15.00 each |
| Tools | Suggested Vendor/Website | Approximate Price (USD) | Notes |
| Planting Tool | Handmade - How-To | (Link to Instructions) -Handmade Tools protocols | |
| Planting Template | Handmade | (Link to File)-Tools Folder | |
| Supplies | |||
| Filter Paper (110mm) 09-851C Whatman #1541-110 |
Fisher Scientific | $44.59/100 | (Picture of planting supplies set up) |
| 1.5㎕ Microcentrifuge tubes # 1615-5599 | USA Scientific | $12.95 | |
| Compact PCR Tube Rack #2300-9602 | USA Scientific | $29.25 |