Preventing contamination in PCR assays

 

Solution

 

  1. Maintain separate areas, dedicated equipment, and supplies for:

 

 

  1. Do not bring amplified PCR products into the PCR setup area.

  2. Wear a clean lab coat (not previously worn while handling amplified PCR products or during sample preparation), and clean gloves when preparing samples.

  3. Change gloves when contamination is suspected.

  4. Open and close all sample tubes and reaction plates carefully. Do not splash or spray PCR samples.

  5. Keep reactions and components capped whenever possible.

  6. Use positive-displacement or aerosol-resistant pipette tips.

  7. Clean the surfaces, lab equipment and pipettes with Ambion® DNAZap solution to degrade contaminating DNA and RNA at the level of PCR sensitivity. The effect of cleaning with DNAZap™ solution is shown in the figures below.

  8. Minimize opening of PCR tubes post-amplification. Real-time PCR systems eliminate the need to open reaction tubes for analysis.

  9. If NTC contamination is encountered:

 

Effect of DNAZap™ solution on plasmid DNA. Plasmid DNA (0.5 µg) was placed in each of two microcentrifuge tubes. Ten µL of DNAZap was added to one tube and 10 µL of water was added to the other. The tubes were briefly mixed by vortexing and then were microcentrifuged. The mixtures were electrophoresed in a 1% agarose gel and stained with ethidium bromide.

 

Efficient cleaning of PCR tubes with DNAZap™ Solution. Plasmid DNA (50 ng), containing an insert amplifiable by PCR, were placed in each of two PCR tubes and dried to completion in a vacuum microcentrifuge. One tube was washed with DNAZap (1 x 200 µL) solution while the other was washed with water (5 x 200 µL). Another 5 ng of DNA was placed in a third tube, and PCR was carried out in all three tubes. The PCR products were electrophoresed in a 1% agarose gel and stained with ethidium bromide.