DNA clean-up

Overview
Digestions
pGT4ΔB
Electrophoresis
DNA clean-up
Ligation
Fragment isolation
Competent cells
Transformation
Recombinants
pGTλ3758ΔH
Miniprepping
Blotting
Probe Labeling
Hybridisation
Probe Detection
PCR

DNA from reaction mixtures (e.g. digestions, PCR, filling-in DNA ends using Klenow polymerase) can be cleaned by centrifugation through silica filters. This method is a convenient alternative for the ethanol precipitation which often is the final step in nucleic acid purification.

In certain high-salt solutions, DNA will bind to silica. An ethanol-containing solution is used in a wash step and a low salt solution is used to release the (clean) DNA from the silica.

Shown below is one page of the product manual of the High Pure PCR Purification Kit (Roche Applied Science). It explains the principle of the DNA purification using spin filter tubes (see pictures below).
The PCR Purification kit can be used for efficient clean-up of any DNA of moderate size (100- 10000 bp) from various (not only PCR) reaction mixtures.

In general, the steps in the cleanup of DNA using silica filters are:

1. Mix a high-salt solution (Binding Buffer) in the DNA solution.

2. Apply the mixture onto a silica filter, and centrifuge.
   The DNA is bound to the filter.

3. Add an ethanol containing wash solution (Wash Buffer) onto the silica filter, and centrifuge.
   The DNA is still bound to the filter, everything else is washed away.

4. Add a low-salt solution (Elution Buffer) onto the silica filter, and centrifuge.
   The DNA comes off from the filter. The eluate is a clean DNA solution.

 

	Spin filters (with a white layer of silica  at the bottom of the tube)
	A spin filter in a 2ml collection tube:
	A spin filter in a regular 1.5ml reaction vial

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