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Basic Methods in Cellular and Molecular Biology
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JoVE Educación científica Basic Methods in Cellular and Molecular Biology
Gel Purification
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  • 00:00Descripción
  • 00:39Gel Purification: Basic Principles
  • 01:51The Gel Purification Procedure
  • 04:51Application
  • 06:04Summary
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Gel Purification

English

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Englishالعربية中文NederlandsFrançaisDeutschעבריתItaliano日本語한국어PortuguêsрусскийEspañolTürkçe

Descripción

Gel purification is used to recover DNA fragments after electrophoretic separation. DNA recovery from an agarose gel includes three basic steps: binding, washing and eluting from a silica column. DNA is believed to bind to silica in the presence of high salt via a salt bridge. Following binding, DNA is washed of impurities and eluted under low salt conditions disrupting this interaction.

This video goes through a step-by-step, generalized procedure for cutting out a band from the gel, gel solubilization, purification through binding to a silica column, and elution of purified DNA. In addition, the presentation discusses several tips for ensuring successful gel purification, including the importance of running an agarose gel with a marker or ladder that has DNA of known sizes.

Procedimiento

Gel-purification is a standard procedure performed to recover desired DNA fragments from agarose gels after electrophoretic separation. After dissolving the gel fragment and running it through a specialized filter, this procedure yields DNA freed from impurities such as salts, free nucleotides and enzymes, suitable for downstream applications. The basic principle behind DNA recovery from agarose gel involves a sequence of bind, wash, and elute steps. Once the gel is in solubilizing buffe…

Divulgaciones

No conflicts of interest declared.

Transcripción

Tags

Gel PurificationDNA RecoveryAgarose GelElectrophoretic SeparationSpecialized FilterImpuritiesDownstream ApplicationsBindWashEluteSilica-filterCentrifugationSolubilizing BufferSalt ConcentrationCation Salt BridgeNegative ChargesEthanol WashElutionPurified DNACasting Agarose GelElectrophoresisUV Light VisualizationMolecular Weight Comparison
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JoVE Science Education Database. JoVE Science Education. Gel Purification. JoVE, Cambridge, MA, (2023).

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