Rolling Circle Amplification (RCA)1-3 is a powerful
isothermal nucleic acid amplification technique particularly useful for
amplifying circular DNA constructs, such as plasmids. The process begins
with a DNA template and short DNA primers. Phi29 DNA Polymerase extends
the primers, continuing to synthesize DNA around the circular template
by displacing the previously synthesized DNA strand when encountered.
Unlike Polymerase Chain Reaction, RCA is an isothermal reaction,
eliminating the need for thermal cycling and allowing for ease of
reaction scalability.
Key features of Phi29 DNA polymerase include: 1) high-processivity,
incorporating tens of thousands of nucleotides without dissociating from
the template, 2) strand displacement ability to dislodge the previously
synthesized DNA strand allowing for continuous amplification of the
template and 3) a very low error rate (~1 in 106-107 bases) resulting from inherent 3'→5' proofreading exonuclease activity, ensuring accurate amplification.4,5
The protocol provided with the TempliAMP™ RCA Kit is versatile and can be applied to various types of circular DNA templates:
- a) Amplification of purified DNA.
- b) Direct amplification from bacterial sources containing circular
DNA template of interest, such as plasmid. This protocol eliminates the
need for DNA extraction, allowing for RCA of circular DNA template
directly from colonies on agar plate, glycerol stocks and liquid media
cultures.
Product Performance:
Figure 1. TempliAMP™ RCA reactions were completed
following the recommended protocols using purified plasmid (3.5 ng), a
bacterial colony, liquid culture (2 µL of a 1:10 dilution), and glycerol
stock (2 µL of a 1:10) as input. RCA reaction products were visualized
on a 1% non-denaturing agarose gel. The TempliAMP™ RCA Kit allows for
direct amplification of purified plasmid, bacterial colonies, liquid
cultures, and glycerol stocks.
Figure 2. Microgram quantities of DNA can be
generated from as little as 1 pg input. The TempliAMP™ protocol is
designed for a DNA input from 5 pg to 100 ng.
Figure 3. 5 ng of purified plasmid DNA was used as
input into three commercial RCA kits (T, N, Q) and the TempliAMP™ RCA
Kit from CELLSCRIPT™ (CS). Quadruplicate reactions were completed
following the manufacturers’ protocols. The TempliAMP™ RCA Kit offers
superior DNA yield, averaging 40 µg per reaction.
Materials Supplied:
Important Store at -20°C in a freezer without a defrost cycle. Do not store at –70°C.
|
TempliAMP™ RCA Kit Contents (25 reactions) |
|
Kit Component |
Reagent Volume |
TempliAMP™ Phi29 Enzyme Solution
in 50% glycerol, 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT),
0.1 mM EDTA and 0.1% Triton® X-100. |
65 μl |
|
10X TempliAMP™ Reaction Buffer |
125 μl |
|
25 mM dNTP Solution |
50 μl |
|
500 μM Random Primer Mix |
65 μl |
|
100 mM Dithiothreitol (DTT) |
50 μl |
|
RNase-Free Water |
3 x 1.6 ml |
Random Primer Mix
5’- N N N N N*N*N -3’ where N = any DNA base and
* = Phosphorothioate bond (to prevent degradation by TempliAMP™ Phi29 proofreading activity)
Materials Required, but not Supplied
- AMPure® XP Bead-based Reagent (Beckman Coulter) for DNA clean-up.
- Thermocycler or heat block capable of 95°C incubation.
- 70% ethanol.
- Optional: Materials for agarose gel electrophoresis and visualization.
- Optional: Materials for fluorescence-based DNA quantification.
Terms & Conditions: The Product listed is currently offered by CELLSCRIPT™ for research use only under the defined Terms and Conditions.
FAQs: For answers to Frequently Asked Questions about our products, visit the FAQ library.
Troubleshooting Guide:
|
Symptom |
Solution |
Low or No
rolling circle amplification
product observed |
Too little starting DNA template was used in the reaction.
• Increase amount of input DNA template ≥5 pg. |
DTT reagent has become oxidized making it inactive.
• Repeated freeze-thaw cycles can reduce the activity of DTT. Repeat the RCA reaction with fresh DTT. |
If AMPure XP beads were used for
purification, RCA reaction product was not fully solubilized before
proceeding.
• Repeat the RCA reaction making sure that all of the beads can be
homogeneously dispersed in the solution before proceeding. If the beads
are still clumpy after 30 minutes, continue incubation and mix the tube
every 15 minutes until the beads can be homogeneously dispersed.
|
Inhibitors are present in the input DNA sample.
• Purify the DNA prior to use in RCA to remove the inhibitors. |
Inhibitors are present in the input DNA sample.
• Purify the DNA prior to use in RCA to remove the inhibitors. |
RCA reaction product is
resistant to restriction
enzyme digestion |
Inhibitors are present in the input DNA sample.
• Purify the DNA prior to use in RCA to remove the inhibitors. |
If you cannot find the answer to your problem then please contact us or telephone +44 (0)1954 210 200