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 (Figure 1). 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. To restore 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.

High recovery rates

The NucleoSpin Gel and PCR Clean-up kit showed a high recovery rate for a wide range of fragment sizes, down to 50 bp (Figure 2). DNA fragments of different sizes were purified from a standard PCR buffer using the kit, with elution volumes of 15 μl, 30 μl, and 50 μl. DNA was analyzed and quantified using an Agilent Bioanalyzer 2100.

Figure 2. High recovery rates for PCR cleanup of fragments down to 50 bp.

Consistent performance with different polymerase buffer systems

Recovery rates and efficiency of primer removal using the NucleoSpin Gel and PCR Clean-up kit were shown to be comparable for three different polymerase buffer systems, unlike competitor kits which showed lower recovery and/or inefficient primer removal (Figure 3). A 165-bp PCR fragment was amplified using different DNA polymerase reaction mix formulations (a–c). Additional primers were added and the mixture was purified using competitor kits from Q, S, and R. The elution volume was 25 μl. For analysis, the complete eluate was loaded onto a 1% TAE agarose gel (u: unpurified). In comparison to MN (the NucleoSpin Gel and PCR Clean-up kit), the other kits all showed lower recovery or inefficient removal of primers. The Competitor Q kit showed a comparable recovery rate but inefficient removal of primers.

Reliable sequencing results

50 ng of a 1,730-bp PCR product purified using the NucleoSpin Gel and PCR Clean-up kit was cycle-sequenced with standard BigDye-Terminator chemistry using an ABI PRISM 3130 Genetic Analyzer. The reading length of up to 750 bp was excellent, and up to 900 bp could be analyzed (data not shown). Data kindly provided by Dr. A. Kocyan, Systematic Botany, Ludwig Maximilian University Munich, Germany.

High recovery with low elution volumes

The NucleoSpin Gel and PCR Clean-up kit provides high recovery rates with elution volumes as low as 15 µl (Figure 4). A PCR fragment (782 bp) was purified from a 1% TAE agarose gel according to the standard NucleoSpin Gel and PCR Clean-up kit protocol using different elution volumes as shown. All eluates were adjusted to 25 μl plus 4.5 μl loading dye. For analysis, the mixture was loaded onto a 1% TAE agarose gel. Recovery was estimated against reference samples (Lanes 1–4). Even with an elution volume as low as 15 μl, recoveries of up to 75–100% can be achieved.

Optimal recovery for small fragments

The NucleoSpin Gel and PCR Clean-up kit showed optimal primer removal and high recovery rates for 50-bp and 100-bp PCR fragments (Figure 5). A sample containing 50-bp and 100-bp PCR fragments and a 27-nt primer was purified using the NucleoSpin Gel and PCR Clean-up kit. The eluate was analyzed on a Agilent Bioanalyzer 2100 and compared to the input before purification. The 15-bp peak displays the internal Bioanalyzer marker. The results showed an optimal removal of the 27-nt primer and a very high recovery (>90%) of the 50-bp and 100-bp PCR fragments.

Gel extraction of a wide size range of DNA fragments

The NucleoSpin Gel and PCR Clean-up kit showed high recovery of a wide size range of DNA fragments extracted from agarose gels (Figure 6). DNA fragments of different sizes were purified from a 1% TAE agarose gel using the kit. DNA was analyzed and quantified using an Agilent Bioanalyzer 2100.

• For PCR clean-up: after addition of Binding Buffer NTI, the mixture is applied to the silica membrane. Contaminants are removed by a simple washing step with ethanolic Buffer NT3.

• For gel extraction: the agarose gel slice is dissolved in high-salt Buffer NT and applied to a NucleoSpin Gel and PCR Clean-up Column, followed by centrifugation and a subsequent washing step. Pure DNA is eluted under low ionic strength conditions with slightly alkaline Buffer NE (5 mM Tris/HCl, pH 8.5).