Principle

The NucleoSpin® Gel and PCR Clean-up Midi

The NucleoSpin® Gel and PCR Clean-up Midi procedure is the easiest way to purify DNA fragments from agarose gels as well as for direct purification of PCR products. The kit includes one buffer for both applications, which contains a pH indicator displaying you the correct pH for optimal kit performance. The purification procedure from enzymatic reactions (e.g., PCR) allows fast and easy removal of enzymes, nucleotides, salts, and other impurities. The NucleoSpin® Gel and PCR Clean-up Midi Columns provide convenient performance for PCR clean-up: After addition of Binding Buffer NTI the mixture is applied onto the silica membrane. Contaminations are removed by a simple washing step with ethanolic Buffer NT3. For gel extraction the agarose gel slice is dissolved in high-salt Buffer NTI and applied to a NucleoSpin® Gel and PCR Clean-up Midi Column followed by centrifugation and a subsequent washing step. Pure DNA is finally eluted under low ionic strength conditions with slightly alkaline Buffer NE (5 mM Tris/HCl, pH 8.5).

NucleoSpin® Gel and PCR Clean-up procedure

Binding Buffer NTI with pH indicator

The optimal pH to bind even small DNA fragments to the silica membrane is about 5–6. Binding Buffer NTI is sufficiently buffered to maintain this pH. However, to be sure that the pH is right even for samples with extreme alkaline pH or high buffer concentration, a pH indicator has been added. The pH indicator does not interfere with DNA binding and is completely removed during the purification. The yellow color is also beneficial for gel extractions,making it easy to identify undissolved pieces of agarose.
For restoring the correct conditions add more Buffer NTI or 4 M sodium acetate (NaAc, pH 5.0), or small amounts of hydrochloric acid (HCl) until the color switches back to yellow.

Colors of the indicator

Features

• Two applications in one kit – one buffer with optimal performance for both applications
• High recoveries for fragments down to 50 bp
• Binding buffer with pH indicator
• Separate buffers for single stranded DNA or SDS containing samples available
• Suitable for all gel buffer systems (e.g., TAE, TBE)

 

Technology	Silica-membrane technology
Format	Midi spin columns
Sample material	< 4 mL PCR reaction mixture < 4 g TAE / TBE agarose gel
Fragment size	50 bp–approx. 20 kbp
Typical recovery	70–95%
A260/A280	1.75–1.85
Elution volume	200–400 µL
Preparation time	25 min/6 preps
Binding capacity	75 µg