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# 605CefT - Resting Medium (no selection)

## Author
[leiboffs](https://protocols.io/researchers/70532)  
Oregon State University, College of Agricultural Sciences, Department of Botany and Plant Pathology

## Disclaimer
**DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK**

The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to [protocols.io](https://protocols.io) is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. 

## 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
Nov 06, 2023

## Last Modified
Nov 07, 2023

## Protocol Integer ID
90512

## Funding Acknowledgement
NSF  
Grant ID: IOS-2211435

## Goal/Experiment
This protocol provides guidelines for preparing 605CefT medium, used in the transformation protocol for somatic embryogenesis of B104 immature maize embryos. The focus is on preventing Agrobacterium contamination and encouraging rapid plant growth.

## Abstract
This part of the Leiboff Lab maize transformation protocol aims to transfer embryos from Co-cultivation Medium 562v-MSM to Resting Medium 605CefT to suppress Agrobacterium contamination. This ensures growth and preparation of embryos for further experimental procedures.

Embryos will be transferred scutellum side up after 3 days of infection, using 605CefT for 7 days before moving to 605CefTB. 605CefT includes synthetic auxin (2,4-D) and uses Cefotaxime and Timentin for suppressing Agrobacterium contamination.

### Professional/Scientific Terms and Reagents
- **2,4-D (2,4-Dichlorophenoxyacetic acid):** A synthetic auxin used for promoting callus formation and shoot growth.
- **Cefotaxime and Timentin:** Antibiotics used to control Agrobacterium contamination.
- **Sucrose and D-Glucose:** Sugars that support rapid plant growth.
- **Casein Hydrolysate:** A mixture of amino acids and peptides used as a nitrogen source.
- **Phytagar:** A gelling agent used to solidify the medium.

### Equipment and Vendors
- **pH Meter:** Hanna Instruments
- **Autoclave:** Cord 3112 and 4112
- **Beakers, Stir Bars, and Graduated Cylinders:** Standard lab suppliers

### Alternative Methods
If specific reagents are difficult to source:
- Casein Hydrolysate can be replaced with a similar complex nitrogen source.
- If synthetic auxin (2,4-D) is unavailable, consider using another auxin-like Indole-3-acetic acid (IAA).

## Planning
1. Estimate the volume of 605CefT needed:

    \[
    \text{Volume} = 30 \text{mL} \times \text{Number Plates}
    \]

2. Prepare mixing ingredients:
    - **Retrieve Heat-Stable Ingredients:**
        - 605 Medium
        - Casein Hydrolysate
        - 2,4-D (5 mg/mL)
        - Sucrose
        - D-Glucose
        - Agar, Phyto

3. Equipment set-up:
    - Graduated cylinder
    - Beaker with a stir bar

## Procedure

### Mixing Heat-Stable Ingredients

1. **Clean equipment:** 
    - Rinse stir bar, beaker, and graduated cylinder with MQ H2O.

2. **Prepare solution in beaker:**
    - Add approximately 90% of final volume using MQ H2O to the beaker.
    - Place on a magnetic stir plate.

3. **Add ingredients:** 
    - Use a dry spatula/pipette for each ingredient.
    - Use the table for specific quantities.

    | Ingredient | 100 mL | 200 mL | 300 mL | 600 mL |
    |------------|--------|--------|--------|--------|
    | 605 Medium | 1.1 g  | 2.2 g  | 3.3 g  | 6.6 g  |
    | Casein Hydrolysate | 0.03 g | 0.06 g | 0.09 g | 0.18 g |
    | 2,4-D | 11.5 µL | 23 µL | 34.5 µL | 69 µL |
    | Sucrose | 2.0 g | 4.0 g | 3.0 g | 6.0 g |
    | D-Glucose | 0.06 g | 0.12 g | 0.18 g | 0.36 g |

4. **Adjust pH:**
    - Set pH to 5.7 using 0.1 M KOH, stir solution.

### Bring to Target Volume and Autoclave

1. Bring solution to target volume:
    - Add water, transfer to a graduated cylinder.

2. Add Phytoagar:
    - According to required mass.

3. Autoclave:
    - Loosely cap the bottle, autoclave using 'Liquid' setting for 20-25 min.

### Adding Heat-sensitive Ingredients
1. Retrieve solution from autoclave, cool to 55°C.
2. Add heat-sensitive ingredients: 
    - Use table below for quantities.

    | Ingredient | 100 mL | 200 mL | 300 mL | 600 mL |
    |------------|--------|--------|--------|--------|
    | Dicamba | 120 µL | 240 µL | 360 µL | 720 µL |
    | Silver nitrate | 340 µL | 680 µL | 1020 µL | 2040 µL |
    | Cef | 100 µL | 200 µL | 300 µL | 600 µL |
    | Tim | 33 µL | 67 µL | 100 µL | 200 µL |

### Final Steps

1. Pour media into clean sterile plates (~30 mL/plate).
2. Close plates to solidify in a laminar flow hood.

## Storage
- Store plates upside-down at 4°C for up to 1 week.

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