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Carrier DNA For Yeast Transformation Preparation of high molecular weight single stranded carrier DNA for yeast transformations.
 

Summary: Carrier DNA For Yeast Transformation
Preparation of high molecular weight single stranded carrier DNA for yeast transformations.
1. Dissolve 100 mg DNA in 10 ml TE, pH 8 in a sterile 50 ml plastic tube. Rotate at room
temperature until completely dissolved. The 10 mg/ml solution will be viscous.
Use salmon testes DNA, sodium salt, 100-250 mg (Sigma D-1626).
2. Centrifuge for 5 minutes at 2000 rpm to collect solution at bottom of tube. Place on ice.
3. Sonicate DNA solution in tube with tip sonifier. Sonify water and rinse before use to clean tip.
Adjust sonication to obtain high output (5 W). Do five 1 minute pulses. Chill on ice for 1 minute
in between. The DNA solution should not heat above room temperature.
4. Run 2-4 g of DNA on an agarose minigel along side a DNA ladder. Repeat sonication in 1
minute pulses until most fragments are between 1 and 10 kb, with an average size of about 5 kb.
5. Extract with an equal volume of phenol:CHCl3. Centrifuge for 5 minutes to separate layers.
6. Precipitate DNA. Add 1/10 volume 3 M NaOAc, pH 5 and 2 volumes 100% EtOH. Precipitate at
room temperature for 10 minutes. Centrifuge for 10 minutes at 2000 rpm to collect pellet.
7. Disperse pellet in 70% EtOH. Centrifuge for 10 minutes at 2000 rpm. Remove all 70% EtOH.
8. Dissolve pellet completely in 10 ml sterile TE, pH 8. This may require 1-2 days.
9. Heat denature DNA solution in boiling water bath for 5 minutes. Plunge tube into ice bucket to
cool quickly. Keep on ice. This denaturation step also sterilizes the DNA.
10. Freeze DNA in 1 ml aliquots at -20C.
To use, quickly thaw at 37C and place on ice. Refreeze as soon as possible.

  

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

 

Collections: Biology and Medicine