The overall aim of this experiment is to isolate root tissue samples from plant seedlings grown in 96 well plates for DNA extraction and genotyping. This is accomplished by first germinating seedlings in special 96 well plates that contain agar growth media and have holes in the bottom of each well. The second step is to stack the seedling plate on top of a standard 96 well plate that is filled with water.
Once the roots have grown down into the lower 96 well plate, the stacked plates are placed in a freezing block to freeze the water in the lower plate. The final step is to peel the upper plate away from the lower plate, breaking off the roots, which are frozen in the lower plate. Ultimately, the resulting root tissue samples can be used to extract DNA samples from each individual plant for genotype analysis.
The main advantage of ice cap compared to alternative methods is that it lets you isolate DNA from thousands of plants in a single day. This level of productivity is possible because with ice cap plants are handled 96 at a time rather than the traditional one at a time. Some examples of how icecap can be used include screening large populations of plants to identify rare recombinants between closely linked IDE generating plants that carry a large number of different mapped mutations and the performing marker assisted selection in crop species.
Basically, any experiment that costs for genotyping, large numbers of individual plants can potentially benefit from the icecap vessel. We Originally Developed icecap for use of the rabbit abscess, but we have also had success using it to collect tissue from rice and tomato seedlings. We expect that ice cap will also be effective on other species as long as their seeds are small enough to fit into a 96 wall plate.
First wrap seedling plates in foil and autoclave at the same time. Soak the clear plastic lids for the seedling plates in 95%ethanol after the autoclave cycle has finished, allow the seedling plates and lids to thoroughly dry in a lamina flow hood. Once the plates are fully dry, apply packing tape to the bottom of each seedling plate and firmly seal using a roller.
Then use a microfill microplate dispenser or multichannel pipeter to dispense 450 microliters of molten growth media into the wells of each seedling plate. Cover the plates with plastic lids and allow the growth media to solidify at room temperature. Sprinkle dry seeds onto watman filter paper and cover each seed thoroughly with 95%ethanol.
Then allow the seeds to thoroughly air dry. Next, transfer the seeds to the seedling plates by first touching the flame sealed end of a modified glass pipette to the surface of the agar media. This moistens the tip of the pipette and allows a single seed to easily be picked up and transferred to the agar surface.
Once every well contains a seed cover each seedling plate with a clear plastic lid and seal with micropore surgical tape. Wrap the stacked plates with aluminum foil and store at four degrees Celsius for four days to stratify the seeds and synchronize germination. After four days, place the seedling plates under fluorescent light at 18 to 20 degrees Celsius for further four days.
To initiate germination, assemble the ice cap fountain on a shelf in the plant growth room so that the surface of the ice cap fountain receives direct fluorescent lighting. To assemble. First, place the custom rack structure inside a plastic storage box and then place a cookie sheet on top of this.
Use the leveling nuts to adjust the level of the cookie sheet. Then place a submersible pump in the storage box and attach the output hose from the pump to the side of the cookie sheet using a spring loaded clamp. Next, fill the ice cap fountain with a mixture of three parts distilled water to one part tap water.
Water should be added until the submersible pump is several centimeters below the water level. Mark the final water level in the plastic storage box using lab tape and a marker pen. Prepare root plates by adding one stainless steel ball to each well of a 96 well root plate.
Then dispense 340 microliters of a mixture of three parts. Distilled water to one part, tap water into each well of the root plate. Peel the adhesive film off of the bottom of the seedling plates and remove the clear plastic lids.
Place each seedling plate in a root plate containing a metal ball and water and secure the paired plates together using two elastic bands. Then place the stacked plates into the ice cap fountain. The paired plate should be left in the ice cap fountain for 10 to 14 days.
During this time, maintain the water level in the ice cap fountain by topping up with distilled water. Once the majority of the seedlings have root tissue that has penetrated the root plates, remove the plates from the ice cap fountain in preparation for the harvesting of root tissue. After the plates have been removed from the ice cap fountain, insert three wooden skewers between the wells of the stacked seedling and root plates.
The skewers will raise the nips of the seedling plates out of the wells of the root plates. Allow the stacked ice cap plates with wooden skewers inserted to stand overnight under lights. This incubation allows slight evaporation of the liquid in the root plates, which facilitates the subsequent freezing and plate separation processes.
On the day of tissue harvest, prepare a freezing bath. Place a 96 well metal thermal block in a Pyrex dish with a mixture of dry ice and 95%ethanol. Allow the thermal block to equilibrate to temperature for around 20 minutes.
Place the stacked ice cap plates with wooden skewers onto the chilled thermal block and incubate for five minutes. During this time, the water in the root plate will freeze solid. Remove the stacked ice cap plates from the freezing bath and place them on the lab bench at room temperature, remove the elastic bands and the wooden skewers from the stacked plates.
Firmly press down on top of the seedling plate to crack the plates. Then carefully peel the root plate and the seedling plates apart. Allow the water in the root plates to thaw at room temperature.
Otherwise, thawing can be expedited by incubating the root plates in a thermal block. Set a 25 degrees Celsius while the root plates thaw. The seedling plates may be prepared for storage.
To do this. Seal the lower part of the seedling plate with adhesive tape. Then place a clear plastic lid on top of the seedling plate and secure with micropore tape.
Stack the seedling plates together, wrap an aluminum foil and place arabidopsis seedlings at four degrees Celsius. The seedlings can be stored at these temperatures for several weeks without affecting viability. Ensure that the water in the root plates has completely thawed.
Before performing the DNA extraction procedure, also inspect the plates to determine if any wells have substantially less water than average pipette distilled water into wells that require additional water. Next, pipette 25 microliters of 500 millimolar TRISS 50 millimolar EDTA solution into each well of the root plate. Then seal the wells of the root plates using thermal sealing foil and a heat sealing machine.
Place the sealed root plates on the geno grinder machine with the sealed end of the plates facing down. Shake the plates in the geno grinder for three and a half minutes at 1, 350 strokes per minute. Transfer the plates to a centrifuge with microplate carriers and spin the plates for 10 minutes at 3, 500 RPM at four degrees Celsius to pellet the pulverized root tissue.
Using a multi-channel pipette transfer 20 microliters of supernatant from each well of the root plate to a fresh 96 well plate that contains 80 microliters of milli Q water in each well. This diluted extract contains genomic DNA that can be directly used in the genotyping PCR reaction according to standard protocols. Once the genotype of each seedling has been identified, remove the seedling plates from cold storage and prepare pots with water saturated soil mixture using pressurized air and a rubber hose, apply gentle air pressure to the small hole in the well of the seedling plate to cause the agar plug and seedling to pop out of the plate onto the lab bench.
Make a small hole about the size of the agar plug in the soil. Then place the agar plug in the soil and pack moist soil around the agar plug to secure the seedling in the soil. Place the transplanted seedlings under light at 18 to 22 degrees Celsius.
Cover each pot with a plastic lid for the first two days after transplants. Then subsequently remove the lid and grow the plant using standard growth conditions. This image illustrates Arabidopsis seedlings that were grown in the ice cap fountain for two weeks and are ready for tissue harvest.
Each of the two wells contains an arabidopsis seedling. This side view of the same two wells of the root plate shows the roots twisting around the inner surface of the wells One mastered. This technique can be used to process up to 40 icecap plates in an eight hour day.
This allows one scientist to produce DNA preps from over 3, 800 individual seedlings in a single day While attempting this procedure, it's important to remember to transfer the soothing plates into the ice cap fountain as soon as the seedlings germinate. Check the plates daily so you don't miss the correct data transfer. Good luck with your experiments.
