One Caution: qPCR is subject to the same contamination concerns as end-point PCR, but those concerns are magnified because the technique is so sensitive. Avoiding contamination is very important in order to generate trustworthy qPCR results. Tips to help you succeed with qPCR: 1. Use aerosol-resistant pipette tips, and have designated pipettors and tips for pre- and post-amplification steps. 2. Wear gloves. Change them frequently. 3. Have designated areas for pre- and post-amplification work. 4. Use “master mixes” to minimize variability. A master mix is a ready-to-use mixture of the reaction components (excluding primers and sample) that you create for multiple reactions. Because you pipet larger volumes to make the master mix, and all of the reactions get their components from the same master mix, you reduce variability from reaction to reaction. 5. Dispense primers into aliquots to minimize freeze-thaw cycles and the possibility of introducing contaminants into a primer stock. Monitor Amplification in Real Time with qPCR 300ng 100ng 10ng 0 1 0 1ng 100pg 0 10pg 1pg 100fg 10fg 00 0f Real-time or quantitative PCR (qPCR) monitors PCR amplification as it occurs, enabling you to measure the starting material in your reaction. Data are presented graphically rather than as bands on a gel. Choose the Real-time qPCR Kit Best Suited to Your Research » enews | November 2015 10302TB Pagina 10
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Labnet Oy - Promega's Monthly online Magazine - November 2015 edition Lees publicatie 101Home