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NucleoSpin® Gel and PCR Clean-up

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The NucleoSpin® Gel and PCR Clean-up 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. 


PCR clean-up and gel extraction – the two-in-one kit with optimized recovery and elution volume

 

• Two applications in one kit – one buffer with optimal performance for both applications
• High recoveries for fragments down to 50 bp
• Minimized elution volume of 15 μL – highly concentrated DNA
• 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)

Characterization:

PCR clean-up and gel extraction – the two-in-one kit with optimized recovery and elution volume

 

• Two applications in one kit – one buffer with optimal performance for both applications
• High recoveries for fragments down to 50 bp
• Minimized elution volume of 15 μL – highly concentrated DNA
• 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)

Applications
• Purification of PCR products
• Extraction of DNA from agarose gels
• DNA extraction from polyacrylamide gels
• Clean-up of single stranded DNA and RNA extraction from agarose gels (Binding Buffer NTC required, not included in the kit, see “Ordering information”)
• Clean-up of SDS-containing samples (Binding Buffer NTB required, not included in the
kit, see “Ordering information”)
• Typical downstream applications: cloning, sequencing, PCR, restriction analysis
• NucleoSpin® Gel and PCR Clean-up ensures complete removal of contaminations such as:
– nucleotides, primers
– enzymes
– mineral oil
– PCR additives (e.g., salts, betaine, DMSO)
– detergents (e.g., Tween 20, Triton X-100)
– dyes (e.g., ethidiumbromide, crystal violet, Stain G, Midori Green, Roti®-Safe GelStain, DNA SafeStain)

 

 


Principle / Procedure

The NucleoSpin® Gel and PCR Clean-up 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 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 NT and applied to a NucleoSpin® Gel and PCR Clean-up 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 indicatorThe 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

 

 

 

 

 


Technical Data:

Application data

NucleoSpin® Gel and PCR Clean-up covers all polymerase buffer systems A PCR fragment with a size of 165 bp 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 was performed with 25 μL. For analysis the complete eluate was loaded onto a 1 % TAE agarose gel (u: unpurified).
In comparison to MN (NucleoSpin® Gel and PCR Clean-up) all other kits show lower recovery or inefficient removal of primers.
Please note that Q shows a comparable recovery rate but inefficient removal of primers!

Reliable sequencing results
50 ng of a 1730 bp PCR product purified using NucleoSpin® Gel and PCR Clean-up have been cycle-sequenced with standard BigDye™-Terminator chemistry using an ABI PRISM 3130 Genetic Analyzer.The reading length up to 750 bp was excellent, up to 900 bp could be analyzed.
Data kindly provided by Dr. A. Kocyan, Systematic Botany, Ludwig Maximilian University Munich, Germany 

Click for downloading
the sequencing profile
as pdf file

High recovery with low elution volumes
A PCR fragment (782 bp) was purified from a 1% TAE agarose gel according to the standard protocol of NucleoSpin® Gel and PCR Clean-up 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 (lane 1–4).Even with an elution volume down to 15 μL recovery of up to 75–100 % can be achieved.

High recovery of up to 90 %
PCR clean-up
DNA Fragments of different sizes were purified from standard PCR buffer using NucleoSpin® Gel and PCR Clean-up and eluted in different volumes (15 µL, 30 µL, 50 µL). DNA quantification was analyzed by Agilent Bioanalyser 2100.
Gel extraction
DNA Fragments of different sizes were purified from 1 % TAE agarose gel by NucleoSpin® Gel and PCR Clean-up. DNA quantification was analyzed by Agilent Bioanalyser 2100. Fragments of 10 to 20 kbp will be recovered with a rate of 50–30 %.

Optimal recovery for small fragments
A sample containing PCR fragments of 50, 100 bp and a 27 nt primer was purified by NucleoSpin® Gel and PCR Clean-up. The eluate was analyzed on a Agilent Bioanalyser 2100 and compared to the input before purification. The 15 bp peak displays the internal Bioanalzer marker. The result show an optimal removal of the 27 nt primer and a very high recovery ( > 90 %) of the 50 and 100 bp PCR fragment. red graph: sample before purification; blue graph: eluate after purification.


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