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DNA Amplification using Illustra PuReTaq Ready-To-Go PCR Beads

PuReTaq Ready-To-Go PCR Beads from Cytiva contain all the necessary reagents (except primer, template, and water) for 25 μL PCR amplifications. The beads are available predispensed into 0.2 mL thin-walled multiwell plates (sufficient for 96 reactions), 0.2 mL thin-walled tubes, or 0.5 mL PCR tubes.

PuReTaq PCR Beads have passed rigorous quality tests to ensure the lowest possible levels of contaminating prokaryotic and eukaryotic nucleic acids.

A protocol for use of Ready-To-Go PuReTaq PCR Beads is provided below.

When performing PCR, exercise extreme care to prevent contamination by nucleic acids. Always use sterile filter pipette tips and microcentrifuge tubes, and avoid carry-over contamination of stock solutions.

For most standard, three-step PCR reactions, 35 cycles results in a 105- to 109-fold amplification of the target sequence. The yield of PCR product may be increased by increasing the number of cycles to 45. However, an increased number of cycles may also produce spurious bands and increased background. The duration of each step when using a “rapid cycler” such as a PerkinElmer™ 9600 thermal cycler (or equivalent) should be approximately half the time as when using a PerkinElmer 480 thermal cycler (or equivalent).

Materials

Each PuReTaq bead contains stabilizers, BSA, dATP, dCTP, dGTP, dTTP, ~2.5 units of PuReTaq DNA polymerase, and reaction buffer. When a bead is reconstituted to a 25 μL final volume, the concentration of each dNTP is 200 μM in 10 mM Tris-HCl (pH 9.0 at room temperature), 50 mM KCl, and 1.5 mM MgCl2.

Template-specific primers

Thermal cycler Mineral oil (if required for your thermal cycler)

Optional: 10 mM MgCl2 (if desired to increase final Mg2+ concentration above 1.5 mM)

Advance preparation

Prepare the PCR beads as follows: Remove the desired quantity of tubes from the foil pouch. Examine these tubes to verify that a bead is visible at the bottom of each tube. If necessary, gently tap the tube against a hard surface to force each bead to the bottom of the tube. Place the tubes into a container that allows easy access during your experiment.

Protocol

1. Add template DNA, primers, and water to bead

For each reaction, add template DNA, template-specific primers, and water to a PCR bead. Optional: Add additional MgCl2 (see Table 7.2, below).

2. Snap caps onto tubes. Mix.

Flick the tube, vortex gently, and centrifuge briefly.

3. Place tubes on ice or in cold block

4. Perform thermal cycling

Add mineral oil if needed for your thermal cycler and replace caps. Perform thermal cycling according to the instructions for your thermal cycler.

The optimal cycling profile for a given PCR system and thermal cycler will vary and must be determined empirically. Cycle number can range from 20 to 40 depending on the desired yield of product. Thermal cycling results and product yield can vary with cycle conditions and thermal cycler used. Read the instructions provided with your thermal cycler and optimize reaction conditions accordingly.

When each PCR bead is rehydrated in a reaction volume of 25 μL, the mixture will contain 1.5 mM MgCl2. If a higher concentration of Mg2+ is desired, Table 7.2 can be used to determine the volume of a sterile 10 mM MgCl2 solution that should be added to increase the Mg2+ concentration of the reaction. If MgCl2 is added to the reaction, decrease the amount of water added to the reaction to maintain a final reaction volume of 25 μL.

Table 7.2Adjusting the reaction concentration of Mg2+ in PuReTaq PCR Bead reactions.
Materials
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