Optimization of a glass fiber membrane based column for improved in-house pcr DNA purification
Column-based PCR purification is commonly used in laboratories to purify PCR amplicons for various downstream applications including sequencing and cloning. The commercially available products such as the Qiagen PCR DNA purification kit, which comes with GMP are designed intended for research usa...
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| Main Author: | |
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| Format: | Monograph |
| Language: | en |
| Published: |
Universiti Sains Malaysia
2025
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| Subjects: | |
| Online Access: | http://eprints.usm.my/63383/1/Muhammad%20Hafiz%20Zaidi-E.pdf http://eprints.usm.my/63383/ |
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| Summary: | Column-based PCR purification is commonly used in laboratories to purify PCR
amplicons for various downstream applications including sequencing and cloning. The
commercially available products such as the Qiagen PCR DNA purification kit, which
comes with GMP are designed intended for research usage. For manpower training or
academic education, a less expensive kit with comparable performance could be a better
option. An in-house self-assembled PCR purification kit using a glass fiber membrane
was developed in the present study, while the QIAGEN kit was used as the reference kit.
For optimization, columns with different membrane pore sizes (0.22 μm, 0.45 μm, and
0.80 μm) and membrane layers (1, 2, and 6) were assembled and tested. The yield and
DNA purities were analyzed via bio-photometer and compared. For results, columns
utilizing GF membrane of a smaller pore size i.e. 0.22 um showed the highest DNA yield.
DNA membrane with 6 layers also retains the highest DNA yield. In the comparative
study, GF based column showed higher performance than the in-house silica membrane
and a comparable DNA yield to the QIAGEN kits. All kits showed good DNA purities
for A260/A280 purities, but not for A260/A230 purities. Nonetheless, the DNA bands
showed to be good in agarose gel DNA electrophoresis analysis. This study suggests that
optimized in-house glass fiber membranes offer a viable and affordable alternative for
PCR DNA purification, bridging the cost-efficiency gap for resource-limited laboratories |
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