Tips and Techniques

Working with PCR Products

Before executing this protocol, become familiar with the polymerase chain reaction (PCR) process and managing PCR amplicon products. The following section provides general guidelines for working with PCR products, but the following guidelines are not all-inclusive. Refer to the site prep documentation for your instrument for more information.

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PCR by design creates many amplicons, which are copies of the input material. PCR products can contaminate reagents, instruments, and samples, delaying normal operations and causing inaccurate results.

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Maintain two separate workspaces for pre-PCR and post-PCR work. Ideally these spaces are physically separated. Perform all work before a PCR amplification in the pre-PCR workspace, and then transfer the sample to the post-PCR workspace for further processing. No material from the post-PCR workspace returns to the pre-PCR workspace.

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To avoid carryover contamination, do not share equipment and supplies between pre-PCR and post-PCR processes. Dedicate a separate set of equipment and supplies in each area.

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If you cannot maintain two separate workspaces for pre-PCR and post-PCR work, keep two separate sets of consumable materials and maintain strict workplace sterility. Make sure all benches and equipment are regularly cleaned with a bleach solution or a commercial DNA degrader to reduce contaminating amplicons. Always wear clean gloves.

Centrifuge Steps

Unless stated otherwise, complete all centrifuge steps with a benchtop minifuge using speeds of 2000 × g or less.

Always use the power button to turn the benchtop minifuge off for a gradual stop that ensures well packed beads.

Thermal Cycler Lid Pressure

Follow the manufacturer instructions for using PCR tube strips with your brand of thermal cycler. Thermal cyclers with excessive lid pressure can cause the 8-tube strip to collapse during whole transcriptome amplification or sample index PCR when only one is used. For brands that do not include plastic frame supports, use empty tube strips as braces surrounding both sides of the 8-tube strip to help balance the lid pressure.