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Related Experiment Videos

Size fractionation using sucrose gradients.

J H Weis1, T Quertermous

  • 1Harvard Medical School, Boston, Massachusetts, USA.

Current Protocols in Molecular Biology
|February 12, 2008
PubMed
Summary
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This study details using linear sucrose gradients to separate DNA fragments by size, aiding in the creation of various genomic libraries. This method offers good resolution for DNA fragments ranging from 5 to 60 kilobases.

Area of Science:

  • Molecular Biology
  • Genomics

Background:

  • DNA fragment size influences migration rate through linear sucrose gradients.
  • Sucrose gradient centrifugation is a valuable technique for DNA fragment separation and purification.

Purpose of the Study:

  • To describe a method for fractionating DNA fragments using linear sucrose gradients.
  • To provide protocols for preparing genomic DNA libraries through size-based fractionation.

Main Methods:

  • Separation of DNA fragments based on size via linear sucrose gradient centrifugation.
  • Protocols for partial and complete genomic DNA digestion using enzymes.
  • Purification of bacteriophage lambda vector arms.

Main Results:

  • Achieved good resolution for DNA fragments between 5 and 60 kilobases (kb).

Related Experiment Videos

  • Demonstrated the utility of sucrose gradients for purifying bacteriophage lambda vector arms.
  • Enabled fractionation of partially digested genomic DNA for cosmid or bacteriophage libraries and completely digested DNA for subgenomic libraries.

    • Conclusions:

    • Linear sucrose gradient centrifugation is an effective method for size-dependent DNA fractionation.
    • The described protocols facilitate the construction of diverse genomic libraries.
    • This technique is crucial for molecular cloning and genomic research.