BACTERIA & PHAGE Templates

Since 1999, GENEWIZ has streamlined the Sanger sequencing process to facilitate genomic research. Our rolling circle amplification (RCA) service allows you to sequence bacterial clones or phage samples without the need for plasmid preparations. RCA generates DNA for sequencing by random hexamer priming of circular templates. GENEWIZ RCA protocols can handle sequencing projects of any size, and are optimized to produce high quality reads from both bacterial clones (colonies and glycerol stocks) and phage samples (plaques or supernatant). RCA is also extremely effective when low amounts of DNA starting material are available (e.g. low copy plasmids).

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Need help choosing the right E. Coli strain for your cloning or protein expression project?

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Download the "Competent Cells Selection Guide" for tips on picking the best protocol for making competent cells as well as the proper strain for your particular experiment.

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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.

The RCA Process

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After lysis, bacterial samples are combined with dNTPs, polymerase, and random hexamers. The hexamers anneal to the circular template, and replication initiates at many sites simultaneously.
Polymerase is utilized due to its high stability, processivity, and high fidelity. This allows prolonged strand displacement over multiple rounds of amplification.
As the newly synthesized strands are displaced, a cascade of priming events facilitates exponential amplification of the template.
Upon completion the sample is ready for Sanger sequencing.

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.

FEATURES & BENEFITS

Time savings and cost savings - no need to grow culture and prepare DNA
>600 bases of Phred20 reads
Free GENEWIZ Universal Primers
Project design and consultation
Analysis using ABI Prism 3730xl DNA analyzers
Fast turnaround
GENEWIZ Online Ordering and Data Retrieval System - 24/7 web-based
Customized troubleshooting

APPLICATIONS

shRNA cloning and library screening; Eliminate plasmid preps and sequence colonies directly.

DNA methylation analysis: Save time & obtain high-quality reads by cloning PCR products directly and sequencing the bacterial colonies.

Antibody phage display library validation: Direct phage sequencing for faster screening and validation.

SAMPLE SUBMISSION OPTIONS

Sample Submission Options

Glycerol Stock* Send 1.5 ml tubes or 96-well plates; samples must be shipped on dry ice. Please label with media type.
Bacterial Colonies* 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.
Phage Supernatant** Please submit 50 ul of phage supernatant in 8-strip PCR tubes or 1.5 ml microcentrifuge tubes clearly labeled with sample name.
Phage Plaque 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: Expression vectors may or may not be suitable for the RCA protocol.

**Please note: RCA is available for circular phage only; linear phage genomes will not amplify by this method.

Please visit our Sample Submission Guidelines for bacteria and phage templates for additional details.


HOW TO ORDER

*Samples must arrive at the GENEWIZ New Jersey laboratory before 10:00 am EST to qualify for Same Day service.  Note that direct-sequencing templates are not available for our Same Day service.



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