Oct 23, 2022
Growing Arabidopsis on nutrient-rich media
- 1University of Pennsylvania
Protocol Citation: Diep R Ganguly 2022. Growing Arabidopsis on nutrient-rich media. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6917dlqe/v1
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: October 21, 2022
Last Modified: October 23, 2022
Protocol Integer ID: 71672
Abstract
Notes on growing Arabidopsis seedlings on nutrient-rich media in plates.
Prepare media
Prepare media
Select and prepare autoclaved media of choice (see recipes below). Make sure to pH before adding gelling agent.
| A | B | |
| MS basal salts | 2.15 – 4.33 g/L | |
| Sucrose | 10 - 30 g/L | |
| Vitamins (1000X) (optional) | 1 mL/L | |
| MES (optional) | 0.1% (w/v) | |
| KOH | pH 5.8 | |
| Agar/Phytoblend | 8 g/L |
MS Media (Murashige & Skoog, 1962)
| A | B | |
| Gamborg’s B-5 basal medium | 3.21 g/L | |
| Sucrose | 10 - 30 g/L | |
| KOH | pH 5.8 | |
| Agar/Phytoblend | 8 g/L |
Gamborg B-5 Media (Gamborg et al 1968)
| A | B | |
| Kanamycin | 30 - 50 uM | |
| Basta (Glufosinate Ammonium) | 20 - 50 uM |
Supplement for transgenic selection - add to cooled autoclaved media immediately prior to pouring plates.
Pour room temperature media into plates in a biosafety cabinet - keep everything sterile! Allow media to cool and solidify, then package and store at 4 °C.
Surface sterilize seed
Surface sterilize seed
Collect small aliquots of desired Arabidopsis seeds into clean microfuge tubes.
Add 500 uL of a solution made of 40% (v/v) household bleach, and 0.01% (v/v) Tween20 dissolved in distilled water and incubate for 5 minutes.
Remove supernatant, then rinse seeds with 700 uL of 70% ethanol incubating for 5-10 minutes. If you did not perform bleach sterilization step, repeat ethanol wash an additional 1-2 times.
Remove supernatant then rinse with distilled water at least 3 times.
After final rinse, plate seeds out on media plates in sterile environment (e.g. BioSafety cabinet). Seeds can be stored at 4 °C for up to 1 month (maximum).