DNA纯化和浓缩新方法:超滤

来源: 默克公司上海办事处   2010-11-11   访问量:7190
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Amicon Ultra-0.5 mL Centrifugal Filters for DNA Purification and Concentration

操作示意图:

With their vertical membrane design, Amicon Ultra 0.5 mL filters deliver great performance and lower spin times

  • Concentrates 500 µL down to 15 µL
  • Concentration factor 25 x to 30 x
  • High sample recovery (greater than 90% of dilute starting solution)
  • Fast processing time - 10 to 30 minutes
  • Consistent recoveries thanks to the reverse spin capability
  • Reverse spin capability provides consistent recoveries

Amicon05_ReverseSpin-71[83660-ALL].jpg

Applications

  • Concentration of biological samples containing antigens, antibodies, enzymes, nucleic acids, or microorganisms
  • Purification of macromolecular components found in tissue culture extracts or cell lysates, primer removal, linkers, or macromelecular labels from a reaction mix, and protein removal prior to HPLC
  • Desalting, buffer exchange, and protein dialysis

Performance – DNA Concentration

The Amicon Ultra-0.5 device provides the best balance between recovery and spin time for double-stranded DNA for base pairs ranging from 137 to 1159. To achieve maximum PCR product recovery and primer removal with primers greater than 20 bases, one or two additional spins with Tris-EDTA (TE) buffer are recommended.

Typical Recovery of Nucleotides from Amicon Ultra-0.5 30K Devices
PCR Product (base pairs) PCR Primer (bases) PCR Recovery (%) PCR Primer Removal (%) TE Washes (number)
137 10 ≥95 ≥90 0
20 ≥90 ≥85 1
48 ≥90 ≥75 2
301 10 ≥90 ≥90 0
20 ≥85 ≥90 1
48 ≥90 ≥80 2
648 10 ≥95 ≥90 0
20 ≥90 ≥90 1
48 ≥95 ≥90 2
1159 10 ≥90 ≥90 0
20 ≥90 ≥95 1
48 ≥95 ≥95 2
Spin conditions: 40º fixed angle rotor, 14,000 x g, room temperature, 500 µL starting volume, 20-30 µL final volume, 10 minute spin, n=12.

Desalting or Diafiltration

Desalting, buffer exchange or diafiltration are important methods for removing salts or solvents in solutions containing biomolecules. The removal of salts or the exchange of buffers can be accomplished in the Amicon Ultra-0.5 device by concentrating the sample, then reconstituting the concentrate to the original sample volume with any desired solvent. The process of "washing out" can be repeated until the concentration of the contaminating microsolute has been sufficiently reduced. See example below.

Amicon05_Desalting-71[83650-ALL].jpg
 
 

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