RCA Service
This service is the fastest way for you to obtain sequence information from a plasmid clone, while saving the time, effort and expense of performing plasmid preps. You can go from colony to sequence in 24 hours.
RCA is short for Rolling Circle Amplification* and is an in vitro process whereby circular DNA from a variety of sources can be amplified to produce high quality sequencing templates. Suitable input DNA includes partially purified or dilute DNA preps, liquid bacterial cultures or bacterial colonies. Amplified templates can be directly sequenced without further purification.
RCA has been widely utilized to amplify DNA for a variety of purposes, including sequencing template preparation.
In RCA, DNA synthesis from a circular DNA (primed by random hexamers and catalyzed by Phi29 DNA polymerase) produces single-strand, linear concatenated copies of the circular sequence. This primary product subsequently acts as template for further DNA synthesis with the products, in turn, also serving as templates for further synthesis. The combined result of this process is exponential amplification of the original circular DNA. Please see Figure 1.
Figure 1. RCA Replicating DNA
Sequetech will, using RCA, amplify and sequence your templates. If requested, we will also pick colonies to serve as a source of your clones.
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Suitable starting material for RCA includes circular DNA or host cells containing plasmids to be sequenced. Any circular DNA (including both single and double stranded) can be amplified.
Partially Purified or Dilute Plasmid Preps - Please supply at least 1 µl of DNA at a concentration of at least 10 ng/µl.
Liquid Cultures - We require at least 100 µl of LB grown culture. (Rich broths, such as Terrific Broth or 2xYT are problematical and should definitely be avoided.)
Single Colonies picked from Agar Plates - Cells should be picked into 10 µl of 0.85% NaCl, being careful to avoid transferring any agar. It is not necessary to transfer the entire colony.
Single Colonies on Agar Plates - Sequetech can pick your colonies for you. Upon request, we can return the picked cells and the original plate. Due to shipping considerations, this service is only recommended for local customers. (Sequetech will pick up your plates and insure that they will not be damaged and that your colonies will not be cross-contaminated.)
Customers do not need to provide primers for template amplification. The random hexamers included in the RCA reagents can prime synthesis from any circular template. However, customers should provide either gene specific sequencing primers or request the free use of one of our 150+ vector primers.
Linearized plasmids or PCR products are not suitable for this technique.
How to ship samples for RCA
Local customers that have samples picked up before 10:00 AM or who drop off samples by 11:00 AM: results will be available the following morning. (If you are requesting a 10AM pickup, you MUST schedule the pickup by 5PM one day prior).
Local customers that have samples picked up after 10:00 AM or who drop off samples after 11:00 AM: results will be available within 48 hours.
Sequences from samples received via FedEx Priority: results will be available the following morning.