Low Throughput Direct Cycle Sequencing of Polymerase Chain Reaction (PCR) Products
Book chapter
Nikolaos Tairis and Zouganelis, G. 2023. Low Throughput Direct Cycle Sequencing of Polymerase Chain Reaction (PCR) Products. in: Scarlett, Gary (ed.) DNA Manipulation and Analysis New York, NY Springer. pp. 195–211
| Authors | Nikolaos Tairis and Zouganelis, G. |
|---|---|
| Editors | Scarlett, Gary |
| Abstract | Polymerase Chain Reaction (PCR) products have been traditionally characterized by cloning and cycle sequencing. However, when quick sequencing data are required, the cloning step may be omitted and PCR products can be sequenced directly. We describe here a sequencing protocol that involves the gold standard Big Dye chemistry in a low throughput format using one of the latest sequencing platforms the ABI Seqstudio. Our cycle sequencing protocol follows the following steps: (1) purification of the PCR product with a spin column-based kit; (2) quality & quantity assessment of the PCR product with the use of spectrophotometry & gel electrophoresis; (3) setup and amplification of the cycle sequencing reaction; (4) Capillary Electrophoresis; (5) Sequence Data Analysis. |
| Keywords | Sanger sequencing; PCR; cloning and cycle sequencing |
| Page range | 195–211 |
| Year | 2023 |
| Book title | DNA Manipulation and Analysis |
| Publisher | Springer |
| Humana | |
| Place of publication | New York, NY |
| Series | Methods in Molecular Biology |
| ISBN | 9781071630037 |
| 9781071630044 | |
| ISSN | 1064-3745 |
| 1940-6029 | |
| Digital Object Identifier (DOI) | https://doi.org/10.1007/978-1-0716-3004-4_15 |
| Web address (URL) | http://dx.doi.org/10.1007/978-1-0716-3004-4_15 |
| Output status | Published |
| Publication dates | Mar 2023 |
| Publication process dates | |
| Deposited | 17 May 2023 |
| Journal | Methods in Molecular Biology |
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