Oct 08, 2021
Botrytis cinerea transformation protocol
This protocol is a draft, published without a DOI.
- 1Universidad Andres Bello Santiago Chile, Instituto Milenio de Biología Integrativa (iBIO)
Protocol Citation: Paulo Canessa 2021. Botrytis cinerea transformation protocol. protocols.io https://protocols.io/view/botrytis-cinerea-transformation-protocol-bywcpxaw
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 08, 2021
Last Modified: October 08, 2021
Protocol Integer ID: 53924
Abstract
Day 1: Fungal growth
Day 1: Fungal growth
18h
18h
Add 10 ml sterile MiliQ water on top of a well-sporulated Botrytis cinerea plate (7-day old PDA-bean plate).
3m
Disaggregate conidia.
2m
Collect conidia and filter using Nytex attached to a flask. You can use the spatula to speed up the process.
2m
Inoculate all collected spores in one flask containing 100 ml of maltose medium (1.5%) for 18 hours.
1m
Incubate at 20º Celsius and 120 rpm for 18 hours.
18h
Day 2: Protoplast generation
Day 2: Protoplast generation
2h 22m
2h 22m
Filtrate de ON culture using Nytex and one 250 ml flask. You can use a sterile spoon to speed up the process.
3m
Wash 4-5 times with KC buffer until completing 50 ml (at room temperature). It is important to remove the media. Collect approximately 2.0 gr. of half-wet mycelium or 1.5 gr. if it is really dry. Transfer to a 100 ml sterile flask in order to prepare protoplasts as indicated below.
5m
Place KC buffer on ice and add filtered-sterilize enzyme mixture solution described below to the “half-wet mycelium”.
5m
Incubate for 2 hours at 28 Celsius and 95 RPM.
2h
Filtrate using Nytex on top of a small 100 ml flask. Wash with KC buffer (on ice). For this procedure, use a total of 50 ml KC buffer.
4m
Centrifuge the filtrate at 4000 rpm (50 ml swinging bucket rotor; Eppendorf 5804 R centrifuge) and 5 min, 4 Celsius. Adjust the speed-up and the speed-down setting of the centrifuge to the minimum.
5m
Wash with KC buffer (50 ml) and centrifuge again as mentioned. Resuspend protoplast in the backflow. Alternatively, remove all KC and then add 500 μl KC.
Take a 1:50 aliquot (4μl:200μl) in order to quantify protoplasts in a Thoma chamber. Adjust the protoplasts to the desire 1x108/ml stock solution.
Day 2: Transformation
Day 2: Transformation
48m
48m
Add 70 μl KC to the genetic construct (three 50 μl purified PCRs in 30 μl total).
1m
Add 100 μl protoplasts to each transformation (from the solution prepared in step 13). Incubate 5 min in ice.
6m
Add 100 μl PEG solution and incubate at RT for 20 min.
20m
Add 500 μl PEG solution and incubate on ice for 10 min.
10m
Add 200 μl KC solution for a final volume of 1ml.
1m
Add 500 μl of protoplasts to 50 ml 45 Celsius SH agar (liquid; using 50 ml sterile disposable tubes). Transfer 10 ml to five Petri dishes. Repeat with the remaining 500 μl of protoplasts to end up with 10 plates per transformation.
10m
Incubate the plates at 20-22 Celsius (12:12 h. photoperiod) for 20 hours.
Day 3: Selection
Day 3: Selection
4d 0h 15m
4d 0h 15m
Add 10 ml SH agar with the selection marker: Hygromycin B (70 μg/ml) or Nourseothricin (140 μg/ml).
15m
Approximately, between 3-7 days, thin (and flat) growing colonies will emerge. Transfer each individual colony to B5-medium + 2% Glucose Petri plates with selection: Hygromycin B 70 μg/ml.
4d