PCR Kleen™ Spin Purification Module

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PCR Kleen Spin Purification Module

Overview

Prepacked Spin Columns
Prepacked Spin Column Selection Guide
  Bio-Spin 6 Micro Bio-Spin 6 Bio-Spin 30 Micro Bio-Spin 30 PCR Kleen
Packed support Special grade Bio-Gel P-6 gel Special grade Bio-Gel P-6 gel Special grade Bio-Gel P-30 gel Special grade Bio-Gel P-30 gel Special grade size exclusion gel
Equilibration buffer 10 mM Tris, pH 7.4 or SSC buffer* 10 mM Tris, pH 7.4 or SSC buffer* 10 mM Tris, pH 7.4 or SSC buffer* 10 mM Tris, pH 7.4 or SSC buffer* 10 mM Tris, 1 mM EDTA, pH 7.0
Applications
Desalting of oligonudeotides >20 bases
Labeling reactions:
removal of unincorporated nucleotides >20 bases or bp from DNA
Removal of primers and primer-dimers from PCR products >200 bp
Buffer exchange (restriction fragments, PCR products, enzyme reactions, sequencing templates)
DNA sequencing reaction mixture cleanup**
Riboprobe cleanup***
Desalting of antibody, enzyme, and protein solutions
Purification of proteins of molecular weight >6, 000
Purification of proteins of molecular weight >40,000
Bed volume 1.1 ml 0.7 ml 1.1 ml 0.7 ml 0.6 ml
Retention and recovery 90% recovery of 20 bases or bp, 99% retention of salts 90% recovery of 20 bases or bp, 99% retention of salts 95% recovery of 22 bases or bp, 98% retention of ddNTPs 95% recovery of 22 bases or bp, 98% retention of ddNTPs 85% recovery of ≥700 bp, 95% retention of primers and primer-dimers
Molecular weight exclusion limit, globular proteins 6,000 6,000 40,000 40,000 8,000,000
Sample volume 50–100 µl 10–75 µl 50–100 µl 10–75 µl 25–100 µl
Centrifuge type Swinging bucket Microcentrifuge Swinging bucket Microcentrifuge Microcentrifuge
Autoclavability Yes Yes Yes Yes Yes

* 150 mM NaCl, 17.5 mM sodium citrate, pH 7.0.
** In Tris buffer.
*** In RNase-free Tris buffer.

Rapid Removal of Primer-dimers

Effective, rapid removal of primer-dimers from PCR reactions. Two PCR reaction mixtures were purified with PCR Kleen spin columns. Lane 1, a mixture of 2 µg of a 660 bp DNA fragment with 100 ng of a 25-mer primer-dimer; lane 3, a mixture of 2 µg of a 660 bp DNA fragment with 500 ng of a 25-mer primer; lane 2, primer-dimer purification; lane 4, primer purification; M, DNA markers. Reaction products and purified samples (10 µl each) were analyzed on a 1.5% agarose gel.

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The PCR Kleen Spin purification module uses size exclusion chromatography to remove unincorporated primers, nucleotides, salts, and enzymes from PCR products. This protocol purifies PCR products using simple spin columns that remove the contaminating molecules by absorbing them into porous beads. Larger molecules, such as PCR products greater than 200 bp, cannot enter the beads and are eluted from the column into a collection tube.

Following the purification step, both the unpurified and the purified PCR products can be resolved side-by-side using agarose gel electrophoresis and stained to visually assess the removal of oligonucleotide primers. Once purified, the PCR product is ligated into the pJet1.2 plasmid vector.

The PCR Kleen Spin purification module is an integral component of the Cloning and Sequencing Explorer Series, where it is used to purify a portion of GAPDH gene that has been amplified. The module can also be purchased separately and used in other experiments to:

  • clean any PCR reaction prior to ligation
  • remove primers prior to subsequent nested PCR reactions
  • remove nucleotides before direct DNA sequencing
Method Size exclusion chromatography in spin column
Applications Removal of nucleic acids <32 bases and other reaction mixture components from PCR products >200 bp
Capacity 25–100 µl
Equilibration buffer 10 mm Tris, 1mM EDTA, pH 7.4
Sample preparation time <4 min
Yield 50–80% recovery
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PCR Kleen™ Spin Purification Module

7326300EDU
Fourth module in Cloning and Sequencing Explorer Series classroom study kit, includes PCR Kleen Spin columns, test tubes, curriculum, for 12 workstations; education use only

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Number Description Options
4006142 Instruction Manual, Quantum Prep PCR Kleen Spin Columns, Biotechnology Explorer, Rev B Click to download
2311 PCR Kleen Spin Columns Product Information Sheet, Rev D Click to download
2950 Comparison of PCR Kleen Spin Columns to Traditional Methods for Purification of PCR Products Prior to Sequencing, Rev A Click to download

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