The 4BB™ TruePrime® RCA Kit uses a novel multiple displacement amplification (MDA) methods based on the combination of the recently discovered DNA primase TthPrimPol and the highly processive and high fidelity Phi29 DNA polymerase to amplify single or double stranded circular DNA molecules by rolling circle amplification (RCA). The strong strand displacement capacity of Phi29 DNA polymerase allows TthPrimPol to generate new primers on the displaced strands that are extended by Phi29 DNA pol, resulting in exponential isothermal DNA amplification. 4BB™ TruePrime® RCA Kit eliminates the need for overnight cell culture and conventional DNA purification.
The starting material for 4BB™ TruePrime® rolling circle amplification can be:
- Purified DNA: Plasmids, cosmids, BACs, M13 clones, etc.
- Bacterial cells transformed with plasmids: colonies picked from agar plates, bacterial cultures or glycerol stocks.
Additional Information
Supply: 4BB™ TruePrime® RCA Kit is supplied in a single box, with sufficient reagents for 100 amplification reactions. The system consists of seven components, including the enzymes and buffers to perform the amplification reactions. Contents are provided in clear, screw cap tubes with color coded lids as referenced in the 4BB™ TruePrime® RCA kit handbook.
Shipping and Storage: 4BB™ TruePrime® RCA kit is shipped on dry ice. Upon receipt, the kit should be stored immediately at -20°C in a non frost free (constant temperature) freezer. If stored correctly the product can be kept for at least 24 months after shipping without displaying any reduction in performance. For longer storage periods we recommend a temperature of -80ºC.
Technical Specifications: 4BB™ TruePrime® RCA kit uses a novel and reliable method to achieve accurate amplification of single or double stranded circular DNA molecules. Dedicated buffers and enzymes deliver microgram quantities of DNA. Typical DNA yields from a TruePrime® RCA kit reaction are above 3µg per 25µl reaction and 3-hour reaction time when starting from 1ng of plasmid DNA (3–10kb). For bigger template molecules (150–200kb) yields are approximately 2µg per 25µl reaction and 3-hour reaction time when starting from 10ng. Yields and kinetics will vary if crude or unquantified samples are amplified. Reactions without input DNA (no template controls) do not produce any amplification products during 3-hour reaction times. The product of TruePrime® RCA reaction is high molecular weight, double stranded concatemers of the circular template. Amplification products can be transformed after digestion with a single site restriction enzyme and religation. Store amplified DNA at 4°C for short term storage or -20°C for long term storage.
TruePrime® RCA kit uses alkaline incubation to allow DNA release and denaturation with very low DNA fragmentation. This results in amplified DNA with high integrity and fragment length.
Quality Control: Each batch of 4BB™ TruePrime® RCA kit is tested against predetermined specifications to ensure consistent product quality. Enzymes used in the kit have been tested separately to ensure adherence to specifications.
Intended Use: 4BB™ TruePrime® RCA kit is intended for molecular biology use only and in vitro use only. This product is not intended for diagnosis, prevention or treatment of a disease in human beings or animals.
Patent: Patent protection for TthPrimPol and methods associated with its use is pending.
Safety information: Safety data sheets are available for download. This material may contain substances or activities that are harmful to human health. It should not be ingested, inhaled or brought into contact with skin, and handled with appropriate care in accordance with the principles of good laboratory practices.
Contents:
| Description | Cap color | 100 reactions |
| Buffer D | Red | 1 x 275µl |
| Buffer N | Translucent | 1 x 275µl |
| Reaction Buffer | Yellow | 1 x 550µl |
| dNTPs | Green | 1 x 275µl |
| H2O | Blue | 1 x 1,500µl |
| Enzyme 1 | Purple | 1 x 280µl |
| Enzyme 2 | Orange | 1 x 80µl |
