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Preparing Polymerase Chain Reaction (PCR) Products for Capillary Sequencing.
Cold Spring Harbor Protocols
|November 3, 2016
Summary
This protocol details preparing amplified DNA products for Sanger sequencing, useful for clone verification. It uses enzymatic cleanup to remove PCR byproducts, enabling direct use in DNA sequencing applications.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Sanger sequencing is a foundational method for DNA analysis.
- Verification of cloned DNA inserts is crucial in molecular biology.
Purpose of the Study:
- To provide a streamlined protocol for preparing amplified DNA products for Sanger sequencing.
- To enable direct use of cleaned PCR products in DNA sequencing.
Main Methods:
- Utilizes polymerase chain reaction (PCR) for DNA amplification.
- Employs enzymatic treatment with exonuclease and shrimp alkaline phosphatase to purify PCR products.
- The protocol can be performed manually with a multichannel pipettor or automated using the Biomek FX Workstation.
Main Results:
- Successfully prepared amplified DNA products suitable for Sanger sequencing.
- Removal of unincorporated primers and deoxynucleotide triphosphates (dNTPs) is achieved.
- The purified products are ready for direct use in capillary DNA sequencing.
Conclusions:
- This protocol offers an efficient method for preparing samples for Sanger DNA sequencing.
- The enzymatic cleanup step is effective in removing PCR contaminants.
- The protocol supports routine molecular biology applications such as clone verification.
