i. Carrier DNA can be frozen after boiling and used 3 or 4
times. If transformation efficiencies begin to decrease with
a batch of boiled carrier DNA it should be boiled again or a
new aliquot used.
ii. The lower concentration of carrier DNA (2 mg/ml) in this protocol eases handling and gives more reproducible results.
iii. In previous protocol versions, a phenol : chloroform extraction was used to ensure maximal transformation efficiencies. This extraction may not be necessary if the DNA is of high enough quality. Test your carrier DNA to determine if extraction is necessary.
The lithium acetate solution is prepared as a 1.0 M stock in distilled de-ionized water (dd water) and filter sterilized. There is no need to titrate this solution, yet the final pH should be between 8.4 - 8.9.
The polyethylene glycol (PEG), MW 3350 (Sigma P3640) is made up to 50% (w/v) with dd water and filter sterilized. For optimal transformation efficiencies, care must be taken to ensure that the PEG solution is at the proper concentration. In addition, it is important to store the PEG in a tightly capped container to prevent evaporation of water and a subsequent increase in PEG concentration. Small variations above or below the PEG concentration optimum in the transformation reaction, which is 33% (w/v), can reduce the production of transformants.
Evaporation of the water from the PEG stock solution will result in an increase in the effective concentration of PEG in the transformation reaction and severely reduce the efficiency.
Plasmid DNA can be prepared by standard protocols; extensive purification is not necessary for yeast transformation. RNA is an effective carrier in the LiAc/SS-DNA/PEG transformation procedure (Schiestl and Gietz, 1989) and does not need to be removed from plasmid DNA preparations before Transformation.
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