PCR-based complete viral genome recovery (Sciuridae)

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Method synopsis

Method using abutting primers designed from contigs to amplify and recover complete circular viral genomes, followed by cloning and Sanger sequencing for verification.

Synthesized from method descriptions across 1 paper using this protocol.

Procedure from a recent peer-reviewed implementation

Steps below were extracted from the most recent peer-reviewed implementation of this protocol in the corpus — Diverse cressdnaviruses and an anellovirus identified in the fecal samples of yellow-bellied marmots (2021), Virology. Implementations in other papers (listed below) may differ.

1. PCR amplification for complete genomes
   15 pairs of abutting primers were designed based on these circular ssDNA virus-like contigs and used to amplify the viral sequences using PCR with Kapa HiFi HotStart DNA polymerase following manufacturer recommended thermal cycling conditions with annealing temperature of 55°C and 60°C
   Quantities: 15 pairs of abutting primersDuration: Thermal cycling with 55°C and 60°C annealingConditions: PCR conditions as per manufacturer
   Equipment: Kapa HiFi HotStart DNA polymerase (Kapa Biosystems, USA)
2. Cloning and sequencing
   Amplicons were resolved on 0.7% agarose gel, excised from gel, purified, ligated with pJET 1.2 vector and transformed into Escherichia coli DH5-alpha competent cells. Purified recombinant plasmids were Sanger sequenced at Macrogen Inc. by primer walking
   Quantities: 0.7% agarose gel concentrationDuration: Not specifiedConditions: Standard molecular cloning conditions
   Equipment: pJET 1.2 vector, Escherichia coli DH5-alpha competent cells, 0.7% agarose gel

Typical Equipment

• Kapa HiFi HotStart DNA polymerase (Kapa Biosystems, USA)
• pJET 1.2 vector
• Escherichia coli DH5-alpha competent cells

Output Measurements

• complete viral genome sequences

Papers Using This Protocol (1)

Species Studied With This Protocol