Yeast Transformation - Super-High Efficiency
Updated 6/29/2016 10:10 AM
Introduction
Materials
- 1M Lithium Acetate (LiOAc). (Filter Sterilised (FS))
- 0.1M LiOAc (FS)
- 50% PEG (3,350 MW) – Prepare with autoclaved dH2O, then filter sterilize. The pH should be near neutral.
- Boiled/Denatured Carrier DNA 10mg/mL salmon sperm DNA, aliquot amount needed and heat at 100°C for 8 minutes, followed by quick chill on ice.
- DMSO
- 5mM CaCl2 (FS)
Procedure
Prepare Competent Cells
- Harvest cells in 50mL conical tubes by centrifugation at 2K rpm for 10 minutes in clinical centrifuge. You will need 2‐10 ODU of cells per transformation. Logarithmic phase cells (OD600 = ~0.5) generally give better efficiencies, so inoculating from a saturated overnight culture and growing for a couple of cell doublings gives better efficiencies.
- Resuspend cells in 5mL 0.1M LiOAc. Adjust volume to ½ original volume of cells.
- Spin down cells at 2K rpm at room temperature for 10 minutes.
- Resuspend cells in a small volume of 0.1M LiOAc at a concentration of 20‐100 ODU per ml. You will be using 100 μl (i.e. 2‐10 total ODU) per transformation.
Two‐Step Transformation
- Aliquot and denature the carrier DNA in 100°C block for 8 minutes, then place on ice for ≥ 5 minutes. For best efficiency, use carrier DNA that is freshly boiled and avoid repeated boiling/cooling cycles.
- Aliquot 100 μl of concentrated competent cells to each microfuge tube. Add 10µL carrier DNA to each sample. Mix briefly by vortexing, then add transformation DNA, and mix again.
- Incubate for 30 minutes at RT.
- Vortex briefly and then add 0.9mL of PEG‐LiOAc‐DMSO mixture to each tube. See Table 1 for recipe. Mix gently, but well.
- Incubate cells in PEG‐LiOAc‐DMSO mixture for 30 minutes at RT or 30°C.
Heat Shock and Plating.
- Place tubes in a 42C water bath or heat block for 14 minutes, mixing gently halfway through.
- Spin down cells in microfuge, 8K rpm for 2 minutes. Discard liquid by decanting or aspirating.
- Wash cells by gently resuspending in 250µL 5mM CaCl2, incubate for exactly 10 minutes.
- Aliquot the entire transformation product onto the correct dropout plates. Spread with sterile glass beads or with a ‘hockey stick’.
- Dump the beads immediately after spreading and let plates dry face up.
- Incubate plates at 30°C for 2 days, face down.
Table 1. General Transformation Parameters
Table1
A A | B B | C C | |
1 1 | Reagent | Ideal final conc. | Ideal voumes for single transformation |
2 2 | 50 % PEG-3350 | approx. 30% | 600 μl |
3 3 | 1 M LiOAc | 0.1 M | 90 μl |
4 4 | DMSO | approx. 10% | 100 μl |
5 5 | Denatured salmon sperm DNA | 10 μl per transformation | 10 μl |
6 6 | DNA in dH2O | variable | 100 ul |
7 7 | Competent cells in 0.1 M LiOAc | 2-10 ODU per transformation | 100 μl |
8 8 | Total volume | approx. 1000 μl |
Comments
wow! It worked! neat stuff.
This protocol doesn't contain volume/weight of plasmid DNA or homology repair template. When would it be added into the reaction?
I have a question, are cells washed after incubation with 5mM CaCl2? or are plated straight away?
Did it work for Pichia pastoris integrative transformation with Zeocin selection ?
Super-high efficiency yeast transformation for introducing large fragments of DNA and multiplexed CRIPSR experiments, from Ben Blount (Ellis Lab). Used as part of the Sc2.0 genome synthesis project to routinely transform ridiculously large fragments of DNA.