A: The TempliAMP™ RCA Kit has the advantage of producing >30 µg of DNA from as little as 5 ng of input DNA in 4 hours. It also offers high fidelity amplification under isothermal incubation conditions (no need for thermal cycling) and can amplify DNA directly from various sources without extraction.
A: The first check point would be to check for yield. RCA products can also be digested with a restriction enzyme and run on a gel to confirm expected size of digest products. The product run on a 1% agarose gel will confirm that amplification occurred as intended. The vast majority of product should run larger than 10 kilobases.
A: Unpurified RCA reaction products can be quantified using fluorometric methods like Qubit™ or PicoGreen®. If the sample is purified from leftover dNTPs and primers, then either fluorometric methods or UV-VIS spectrophotometry (e.g., NanoDrop™) can be used.
A: There are two methods to increase yield: More input DNA can be added to the reaction, or the incubation time can be extended. It should be noted that increasing the incubation time could result in more non-specific amplification.
A: Yes, the reactions must be run at 30°C for optimal results or the yield will be impacted. If the temperature is higher the enzyme falls apart, and if the temperature is lower the reactions run slower.
A: Rolling circle amplification requires circular input template, and amplification with TempliAMP™ RCA Kit will therefore proceed most efficiently on a circular template, resulting in a single long stretch of repeated DNA. However, the kit is also able to amplify linear DNA or genomic DNA.
A: The TempliAMP™ RCA Kit can amplify various types of DNA, including purified circular DNA (both single and double-stranded). It can also amplify DNA directly from sources like liquid media cultures, agar plate colonies, and glycerol stocks, without needing to extract the DNA first.
A: Yes, the Phi29 Enzyme Solution can be inactivated by heating the completed reaction to 65°C for 10 minutes.
A: Many downstream applications would only require a dilution of the RCA product, but it is advised to heat inactivate the enzyme prior to proceeding to downstream applications due to the inherent 3’→5’ proofreading exonuclease activity of Phi29 DNA Polymerase. If purification is required, it is recommended to use AMPure® XP Bead Purification. Note that with longer incubations times it can take a long time for the DNA to go back into solution during the elution step.
A: Due to the inherent 3’→5’ proofreading exonuclease activity of Phi29 DNA Polymerase, primers need to be designed to be exonuclease-resistant by incorporation of phosphorothioate bonds between the three 3’-most nucleotides. Random primers included in the kit are exonuclease-resistant. If the use of specific primers is desired, primers should be designed to bind specifically to the circular DNA template.
