DNA Amplification with DryDrops® PCR UDG Lyophilized PCR Beads
Single Assay PCR Lyophilized Beads are a pre-formulated and pre-dispensed reaction mixture for carrying out polymerase chain reaction (PCR). Uracil-DNA glycosylase excludes amplicon contamination from previous reactions and false-positive results. Each lyophilized bead contains all the necessary components for PCR and real-time PCR with a volume of 25 μL.
The composition of the reaction mixture is optimized to obtain ideal results in terms of processivity and specificity of amplification. Lyophilized beads provide excellent reproducibility between reactions by minimizing pipetting steps and reducing the likelihood of pipetting errors and sample contamination. Each batch of the Single Assay PCR Lyophilized Beads undergoes functional testing to ensure batch-to-batch reproducibility.
The lyophilized format allows storing the PCR mixture for up to 12 months at temperatures up to 4 °C.
Reaction Mixture Composition:
- Hot-Start Taq polymerase;
- Uracil-DNA glycosylase (UDG);
- Mixture of deoxynucleoside triphosphates (including dUTP);
- Optimized PCR buffer (contains 3 mM Mg2+ in 1× of the reaction mixture)
- Cryoprotectants
Hardware Compatibility:
DryDrops® UDG lyophilized beads are compatible with all types of cyclers and can be used for PCR in both amplifiers with classic thermoblocks for PCR tubes and with cartridges.
Possible Applications:
DryDrops are suitable for real-time PCR detection, including quantitative analysis (with fluorescent probes or intercalating dye, e.g., Eva488), as well as for DNA amplification followed by electrophoresis detection.
For cloning tasks and other applications that require further work with the PCR product after amplification, we recommend using our dUTP-free DryDrops® PCR product.
Protocol
! The reaction volume may vary depending on the specific task but should always be a multiple of 25 μL.
- Pre-mix the components of the reaction, except DNA, in a separate test tube according to the table below, based
on (N+0.1N) reactions, where N is the required number of reactions.
Calculation per 25 μL reaction with real-time detection:
Component Volume Note Upstream primer 0.5–1.5 μL of 10 μM solution 5–15 pmol/reaction (final concentration 200–600 nM) Downstream primer 0.5–1.5 μL of 10 μM solution Probe
or
Intercalating dye0.25–0.75 μL of 10 μM solution 2.5–7.5 pmol/reaction (final concentration 100–300 nM) According to the manufacturer's recommendation DNA 2–9 μL Will be added in step 4 separately to each test tube Deionized water Add to a total reaction volume of 24 µL Taking into account the volume of the DNA sample that will be added in step 4. Total volume of the reaction mixture 24 μL When using a different reaction volume, the volumes of the reaction components should be recalculated while maintaining the given proportions Calculation for one reaction with a volume of 25 μl with gel electrophoresis detection:
Component Volume Note Upstream primer 0.5–1.5 μL of 10 μM solution 5–15 pmol/reaction (final concentration 200–600 nM) Downstream primer 0.5–1.5 μL of 10 μM solution DNA 2–9 μL Will be added in step 4 separately to each test tube Deionized water Add to a total reaction volume of 24 µL Taking into account the volume of the DNA sample that will be added in step 4. Total volume of the reaction mixture 24 μL When using a different reaction volume, the volumes of the reaction components should be recalculated while maintaining the given proportions
- Wipe clean tweezers with a 70% ethanol solution and dry. Use tweezers to place one bead into each PCR tube. Important! If you increase the reaction volume, you need to put proportionately more beads in each PCR tube.
- Add 15–22 μL of the prepared reaction mixture to the beads, assuming that after adding DNA (2–9 μL), the total volume without the bead should be 24 μL.
- Using a separate pipette tip, add 2–9 µL of DNA/cDNA sample (total 50–100 ng genomic DNA, 1–100 pg plasmid DNA) into each PCR tube. After adding DNA, the total reaction volume should be 25 µL (one bead contributes 1 µL to the reaction volume). Centrifuge the drops.
- Perform DNA amplification using the given programs (primer annealing temperature is calculated individually
for each pair of primers).
If the annealing temperature of primers is ≥60°C
Stage Temperature Time Number of cycles Activation of HS Taq polymerase 95 °C 5 min 1 Denaturation 95 °C 10 s 40–50 Primer annealing combined with elongation
(Fluorescence detection should be performed at this stage)60–72 °C If the annealing temperature of primers <60°C
Stage Temperature Time Number of cycles Activation of HS Taq polymerase 95 °C 5 min 1 Denaturation 95 °C 10 s 40–50 Primer annealing
(Fluorescence detection should be performed at this stage)55–59 °C 10–15 s Elongation 72°C 15–30 s - In case of using an intercalating dye, it is recommended to melt the amplicon in the range of 60 to 95 °C after amplification to be sure in the absence of non-specific amplification,
- To analyze PCR results using gel electrophoresis, mix amplified samples with gel buffer, add them into the gel wells, and perform electrophoresis.
- If necessary, amplification products can be stored at −20 °C.
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