Difference between revisions of "Yeast Colony Transformations"
From PombEvolution
Jump to navigationJump to search (Created page with "Protocol based on [https://onlinelibrary.wiley.com/doi/full/10.1002/yea.1104 Morita & Takegawa 2004] Fast and convenient transformation protocol for fission yeast. This metho...") |
|||
| Line 1: | Line 1: | ||
Protocol based on [https://onlinelibrary.wiley.com/doi/full/10.1002/yea.1104 Morita & Takegawa 2004] | Protocol based on [https://onlinelibrary.wiley.com/doi/full/10.1002/yea.1104 Morita & Takegawa 2004] | ||
| − | Fast and convenient transformation protocol for fission yeast. This method is not as efficient as the [Yeast Chemical Transformations] or the [Yeast Electoporation Transformation]. The cells are grown on a plate to form a colony that is then scraped of the medium, resuspended in transformation medium and immediately used for transformation. | + | Fast and convenient transformation protocol for fission yeast. This method is not as efficient as the [[Yeast Chemical Transformations]] or the [[Yeast Electoporation Transformation]]. The cells are grown on a plate to form a colony that is then scraped of the medium, resuspended in transformation medium and immediately used for transformation. |
| − | # Suspend cells of the to-be-transformed strain in sterile saline or water | + | # Suspend cells of the to-be-transformed strain in sterile [[saline]] or water |
# Pipette the cells on [[YES]] in 10µl droplets | # Pipette the cells on [[YES]] in 10µl droplets | ||
| − | # | + | # Incubate the plate for 24-48 hours at 32°C to form a fat colony of about 5mm |
| − | # Prepare a tube with 50µl PLATE mixture | + | # Prepare a tube with 50µl [[PLATE]] mixture. |
| − | # Scrape the cells of the plate with a sterile toothpick | + | #* 50% <u>P</u>olyethylene glycol (PEG)‐4000 |
| − | # | + | #* <u>L</u>ithium <u>A</u>cetate 100mM |
| + | #* 10 mM <u>T</u>ris-HCl | ||
| + | #* 1mM <u>E</u>DTA | ||
| + | # Scrape the cells of the plate with a sterile toothpick and suspend in the PLATE | ||
| + | # Add 5µl [[ssDNA]] and mix | ||
| + | # Add plasmid and/or Homologous DNA (about 1 µg) and mix | ||
| + | # Heatshock the mix at 42°C for 15 minutes | ||
| + | # Centrifuge the cells at 1600 x g for 3 min at room temperature (RT). | ||
| + | # Remove supernatant and re-suspend the cells in 1 ml | ||
| + | #*[[EMM-N_-_Minimal_Mating_Medium|EMM-N]] '''or''' | ||
| + | #*[[EMM_-_Edinburgh_Minimal_Medium|EMM]] when working with h<sup>90</sup> or an other homothallic strain | ||
| + | #:Reduce the added supplements (e.g. leucine, adenine etc) to 10% of the standard amount. | ||
| + | # Incubate at RT for ~16h | ||
| + | # Spin down the cells at 1600 x g for 3 min, RT and remove supernatant | ||
| + | # Resuspend cells in 100 μl water and spread them on [[YES - Yeast Extract Supplemented | YES]] plates with appropriate selective agent (1 in 1000 [[Nourseothricin]] (100 μg/μl) or 1 in 500 G418 (50mg/ml)). | ||
| + | # Incubate at 32°C for at least 3 days (but more likely 4 to 5) | ||
Revision as of 12:55, 30 October 2018
Protocol based on Morita & Takegawa 2004
Fast and convenient transformation protocol for fission yeast. This method is not as efficient as the Yeast Chemical Transformations or the Yeast Electoporation Transformation. The cells are grown on a plate to form a colony that is then scraped of the medium, resuspended in transformation medium and immediately used for transformation.
- Suspend cells of the to-be-transformed strain in sterile saline or water
- Pipette the cells on YES in 10µl droplets
- Incubate the plate for 24-48 hours at 32°C to form a fat colony of about 5mm
- Prepare a tube with 50µl PLATE mixture.
- 50% Polyethylene glycol (PEG)‐4000
- Lithium Acetate 100mM
- 10 mM Tris-HCl
- 1mM EDTA
- Scrape the cells of the plate with a sterile toothpick and suspend in the PLATE
- Add 5µl ssDNA and mix
- Add plasmid and/or Homologous DNA (about 1 µg) and mix
- Heatshock the mix at 42°C for 15 minutes
- Centrifuge the cells at 1600 x g for 3 min at room temperature (RT).
- Remove supernatant and re-suspend the cells in 1 ml
- Reduce the added supplements (e.g. leucine, adenine etc) to 10% of the standard amount.
- Incubate at RT for ~16h
- Spin down the cells at 1600 x g for 3 min, RT and remove supernatant
- Resuspend cells in 100 μl water and spread them on YES plates with appropriate selective agent (1 in 1000 Nourseothricin (100 μg/μl) or 1 in 500 G418 (50mg/ml)).
- Incubate at 32°C for at least 3 days (but more likely 4 to 5)
