McClean:Yeast Transformation: Difference between revisions
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'''Polyethylene glycol PEG 50% w/v (Sigma P3640)''' | '''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. | 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. | ||
Revision as of 10:46, 20 July 2011
Overview
Our lab's version of the Geitz lithium-acetate transformation method.
Materials
- 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)
Stock Solutions
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 acetat 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
Day 1
- 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.
Day 2
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
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References
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.
Contact
- Who has experience with this protocol?
or instead, discuss this protocol.
