Jan 06, 2020
High throughput pipette-tip hydroponics for collecting salt stress samples for RNA-seq
- 1Boyce Thompson Institute
Protocol Citation: Magdalena M Julkowska 2020. High throughput pipette-tip hydroponics for collecting salt stress samples for RNA-seq . protocols.io https://dx.doi.org/10.17504/protocols.io.bazeif3e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 06, 2020
Last Modified: January 06, 2020
Protocol Integer ID: 31494
Abstract
Growing on plants in hydroponics can take a lot of space, and thus failure to germinate is a risk that is usually taken into account but results in quite a lot of waste of space. Here, I describe a protocol for high-throughput Arabidopsis germination and early growth. The protocol described here is suitable for growing the Arabidopsis seedling for the 1st two weeks after germination. After this time period the seedlings should be transferred into the larger containers or harvested for the analysis.
Guidelines
The protocol described in here is not describing sterile growth conditions, however the materials should be prepared as cleanly as possible to avoid bacterial & fungal infections.
Materials
MATERIALS
MES, free acid, monohydrateBio Basic Inc.Catalog #MB0341.SIZE.25g
Daishin agarDuchefa BiochemieCatalog #9002-18-0
Potassium hydroxideSigma AldrichCatalog #1050121000
Murashige & Skoog Basal SaltsCaisson LabsCatalog #MSP01-50LT
Before start
Make sure you have a lot of yellow pipette tips and preferably plenty of pipette tip boxes for 1 ml pipette tips.
Prepare seeds
Prepare seeds
1d
Sterilize the Arabidopsis seeds (no more than +/- 400 seeds) in 2ml Eppendorf tube with 1 ml of 50% household bleach (diluted with MQ water) - keep for 10-20 minutes
Wash the seeds with 2 ml of sterile MQ water - 5 to 8 times - preferably in the laminar hood
Incubate the seeds in MQ water in the dark at 4C at least overnight, but not longer than one week, to break the seed dormancy
Prepare pipette studs
Prepare pipette studs
1h
Using the large and sturdy scissors - cut the yellow pipette tip so that you only keep the wider stud - as in the picture
The studed pipette tip - the bottom one is the desired cut of the pipette tip
Prepare Agar medium solution. Per 1 L use:
2.2 g Murashi Skoog nutrients
1 g MES buffer
adjust pH with KOH to 5.8
After adjusting pH add 10 g of Dashin Agar
Autoclave the solution or melt the agar in the microwave.
You can store the nutrient agar solution on your benchtop, at room temperature, for about two weeks, or at 65 C cabinet for up to three days. Before using make sure that the solution is liquid by warming it up in the microwave
Put the pipette studs into a clean PCR plate and add liquid nutrient agar solution so that it covers the bottom part - wait for the solution to solidify.
After the initial solidification of the media in the bottom part of the pipette tip - add more agar to fill the pipette tip
TIP: Make sure that your media forms a meniscus rather than a "bulb" - this will make it easier to place tiny Arabidopsis seeds without them sliding to the side
pipette studs in the final stage of filling them with nutrient agar solution
Put the agar-filled pipette studs into the 1 ml tip box (they should be placed deeper into the box than in "yellow tip box"), fill the box using the liquid medium. Per 1L of liquid medium you need:
2.2 g Murashi Skoog nutrients
1 g MES buffer
adjust pH with KOH to 5.8
The above solution does not have to be autoclaved - unless you have a specific reason to do so.
Using 1 ml pipette tip - drop the Arabidopsis seeds that underwent 24h cold treatment on top of the agar in the pipette studs. Place the pipette tip boxes in the growth chamber set at 18C, 12h light/dark cycle, 60% humidity for the seeds to germinate.
NOTE - it is important that the boxes are covered with the transparent lid - so the agar in the studs won't dry out
Make sure to check on the level of liquid media every day and replace the media completely every 5-7 days
Four days after placing the boxes into the growth room you should observe thegermination of your seeds. I would advise you to "thin out" the seedlings at this stage - so you have only one germinating seedling per agar stud
Arabidopsis seedlings 4 days after germination
The roots should grow out of the agar studs between one and two weeks after germination. The level of media is adjusted as not to cover the studs completely, but rather be just below the plate holding the pipette tip studs
One week old seedlings of Arabidopsis in pipette-tip hydroponics
two weeks old seedlings of Arabidopsis
After two weeks of growth, the seedlings are transferred to larger hydroponics containers with 2L of solution per container
Two weeks old seedlings transferred to the larger Ara-ponics containers
For RNA-seq analysis, we add 100 mM NaCl to the liquid nutrient solution (as described above), when replacing the medium at 3 weeks after germination and harvest the shoot and root tissue to separate tubes for RNA isolation.
SUGGESTION: If you wish to treat your seedlings with salt stress, you could do it at earlier stage, as soon as the root will grow out of the agar stud