The TRAFO Solutions Page

Materials required:

Please Note: You Do Not have to Sonicate the Carrier DNA ANYMORE! WHY? I have done the experiments and shown that that Bigger is Better for Carrier DNA! Follow instruction below.

Single-stranded Carrier DNA (2 mg/ml)

1. Weigh out 200 mgs of high molecular weight DNA (Deoxyribonucleic acid Sodium Salt Type III from Salmon Testes, Sigma D1626) into 100 ml of TE buffer (10 mM Tris-HCl pH 8.0, 1.0 mM EDTA). Disperse the DNA into solution by drawing it up and down repeatedly in a 10 ml pipet. Mix vigorously on a magnetic stirrer for 2-3 hours or until fully dissolved. If convenient, leave the covered solution mixing at this stage overnight in a cold room.
2. Aliquot the DNA and store in a -20^o C freezer.
3. Prior to use, an aliquot should be placed in a boiling water bath for at least 5 min and quickly cooled in an ice water slurry.

   TIP:

   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.

Note on Carrier DNA sterility: We dissolve our carrier DNA into sterile TE and then consider it sterile after the 5 min in a boiling water bath. Have not had problems with this approach!

1.0 M Lithium Acetate Stock Solution

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.

Note : Lithium Acetate can be sterilized in the autoclave for 20 mins liquid cycle.

Polyethylene glycol (PEG 50% w/v)

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.

1. Place 50 gm of polyethylene glycol, MW 3350 (Sigma) in a 150 ml glass beaker and add 35 mls of ddH20.
2. Stir with a magnetic stirring bar until dissolved. This will take about 30 min.
3. Transfer all of the liquid to a 100 ml graduated cylinder. Rinse the beaker with a small amount of distilled water, add this to the graduated cylinder containing the PEG solution, and bring the volume to exactly 100 mls. Mix well by inversion.
4. Filter sterilize using a 0.45 µm filter unit (Nalgene), and store in a securely capped bottle.

NOTE: YOU CAN ALSO AUTOCLAVE PEG TO STERILIZE!!! Its a bit easier.

*Note:

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

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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