Sequencing of Bacteria & Phage by Rolling Circle Amplification (RCA)
RCA was identified in natural systems as a DNA replication mechanism frequently employed in plasmid and viral propagation. When random hexamers are coupled with a strand displacing DNA polymerase, many concatenated template copies are generated with no need for thermocycling (Dean et al., 2001). The synthesized strands are displaced after one round of RCA and are free for additional rounds of primer binding. Exponential amplification is achievable if the RCA reaction contains primers complementary to both template strands.
Note that RCA adds an additional 24 hours to the sequencing turnaround time. RCA is recommended for circular templates less than 15 kb in length.
Dean, Frank B. et al. “Rapid Amplification of Plasmid and Phage DNA Using Phi29 DNA Polymerase and Multiply-Primed Rolling Circle Amplification.” Genome Research 11.6 (2001): 1095–1099.
SAMPLE SUBMISSION OPTIONS
Sample Submission Options
||Send 1.5 ml tubes or 96-well plates; samples must be shipped on dry ice. Please label with media type.
||Array colonies in a grid on the agar plate and circle the colonies requiring sequencing. Numerically label each colony. Note that unnumbered plates will be processed as random pick. If using liquid culture, submit samples in 96-well plate with strip caps. Wrap agar plates in parafilm, and provide a cushion to protect against potential damage.
||Please submit 50 ul of phage supernatant in 8-strip PCR tubes or 1.5 ml microcentrifuge tubes clearly labeled with sample name.
||Circle and number the plaques you submit for sequencing. For transport, wrap agar plates in parafilm and provide cushion to protect against potential damage.
*Please note: RCA protocol is intended for circular templates <10 kb; linear templates will not be amplified sufficiently for direct Sanger sequencing.
Longer circular templates, or circular templates carried in bacterial strains with functional endA may not be reliably amplified, contributing to variable results
Please visit our Sample Submission Guidelines for bacteria and phage templates for additional details.