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 -20o 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:

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

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


Polyethylene glycol (PEG 50% w/v)

 

  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:


Plasmid DNA



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