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Comparison of PCR Kleen Spin Columns to Traditional Methods for Purification of PCR Products Prior to Sequencing, Rev A

ces using commercial gel extraction kits from two other suppliers (suppliers A and B).

Following purification, PCR aliquots were analyzed on a 1.2% TAE agarose gel and compared with the unpurified PCR p roduct (aliquot 4). The DNA concentration of all three purified PCR products was evaluated spectrophotometrically and compa red with the concentration of the unpurified PCR p roduct. The DNA concentrations were : unpurified product , 0.978 g/l; product purified by PCR Kleen spin column, 0.064 g / l; by supplier A gel extraction kit, 0.016 g/l; by supplier B gel extraction kit, 0.014 g/l.

Following sequencing PCR, the resulting nucleotide sequences were analyzed on an ABI PRISM 310 automated sequencer and compared .

Experiment 2. Sequencing Quality of Longer PCR P roduct After Sequencing PCR and Dye Te rminator Removal
In order to evaluate the effectiveness of PCR Kleen spin columns on the quality of longer nucleotide sequences, thre e aliquots of a 750 bp PCR product derived from Borna disease v i rus were purified using PCR Kleen spin columns. After sequencing PCR, the recovered products were purified for sequencing by three diff e rent methods: We tested one classical purification method (p recipitation in the presence of C2H5O H and 0.5 mM MgCl2) in parallel with two modern commercially available dye removal kits from two other suppliers.


Results
Experiment 1
The best yield of DNA was achieved after purification of the PCR product with the PCR Kleen spin column (see figure).

In contrast, both PCR products obtained by gel extraction, using either the protocol of supplier A or B, were only weakly detected by agaros
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