|Line 39:||Line 39:|
Revision as of 13:56, 20 July 2011
Our lab's version of the Geitz lithium-acetate transformation method.
- 1M LiAC
- 100mM LiAC
- 50% w/v PEG MW 3350 (Sigma P3640)
- Sterile H20
- Single-stranded carrier DNA (Sigma D1626, 2.0 mg/ml in TE buffer pH 8.0)
- Appropriate selective plates (SC-URA, YPD+G418, etc)
Polyethylene glycol PEG 50% w/v (Sigma P3640)
Make up 50% w/v with H20 and filter-sterilize with a 0.45uM filter unit (Nalgene). We don't autoclave the PEG. Store in a tightly capped container to avoid evaporation.
Single-stranded carrier DNA (Sigma D1626)
- Weight out 200mg of the DNA into 100ml of TE buffer. Disperse the Dna into solution by drawing it p and dwn repeatedly in a 10-ml pipette. Mix vigorosly on a magnetic stirrer for 2-3 hours or until fully dissolved. Alternatively, leave the covered solution mixing at this stage overnight in a cold rom.
- Aliquot the DNA into 100μL portions and store at -20°C.
- Prior to use, the aliquot should be boiled and then quickly cooled on ice. We use a thermocycler to heat the DNA to 95°C for 25 minutes and then rapidly cool it on ice.
Once the salmon sperm has been boiled it can be freeze-thawed 3 or 4 times before transformation efficiencies begin to decrease. In practice, we boil the DNA before every transformation.
TE Buffer (pH 8.0)
- 10 mM Tris-HCL (pH 8.0)
- 1.0 mM EDTA
1.0M Lithium acetate stock solution (LiAc)
Prepare as a 1.0 M stock in distilled deionized H2O; filter-sterilize. The final pH should be between 8.4 and 8.9
- Inoculate the strain to transform from a single colony into 5mls of YPD in a test tube. Put on the roller drum at 30°C overnight.
- Inoculate 50 ml of YPD with 500 μL of the YPD overnight culture in a 250 ml flask. The 700 µl volume is approximate, and depends on the density of the strain you inoculate.
- Grow in shaking incubator for about 3-5 hours.
- Turn on 42°C water-bath (for heat-shock) if it is not already on.
- Harvest the cells by centrifuging in Eppendorf centrifuge model 5810R at 4000rpm (3130 xg) for 5 min. Resuspend pellet in 25 ml of sterile water by vortexing briefly. Pellet again and then resuspend in 1 ml of 100 mM LiAc.
- Transfer cell suspension to a 1.5 ml eppendorf tube, centrifuge at 3,000 xg for 2 min in an Eppendorf 5418 centrifuge and discard supernatant by removing it with a pipette.
- Add 400 µl 100 mM LiAc and resuspend cells by pipetting up and down. Aliquot 50 μL into 1.5 ml tubes (1 for each transformation). Pellet cells (3,000 xg for 2 min) and remove supernatant by aspiration.
- Add 300 μL T mix to each eppendorf tube of cells. Per one transformation reaction add IN ORDER:
- 240 μL 50% PEG 3350
- 35 μL 1.0 M lithium acetate
- 25 μL 2 mg/ml sssDNA
- 50 μL sterile H20 and 20 μL of DNA (Note: You are aiming for a final concentration between 0.1-10 μg for plasmid DNA. Adjust your DNA and water amounts to add 70 μL of volume total)
- Vortex to resuspend cells.
- Incubate for 30 minutes at 30°C.
- Incubate tubes in a water bath at 42°C for 20-25 (up to 40) min. The time may need to optimized for your strain and transformation conditions.
- Microfuge at 3,000 xg for 15s, and remove transformation mix with a micropipette.
- Add 200 µL of sterile water to each tube and resuspend cells by pipetting it up and down as gently as possible if high efficiency is important.
- Plate 150 µl of sterile water and add 20 µl cell suspension in one selection plate #1.
- Plate the remaining 180 µl in selection plate #2
- Incubate at 30 °C. Colonies should appear after 2-4 days.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- Anecdotal observations that might be of use to others can also be posted here.
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
Gietz, R.D. and R.A. Woods. (2002) TRANSFORMATION OF YEAST BY THE Liac/SS CARRIER DNA/PEG METHOD. Methods in Enzymology 350: 87-96.
- Who has experience with this protocol?
or instead, discuss this protocol.