Maximizing PCR and RT-PCR Success

6 PCR Brochure 10/2016 Fresh QIAGEN Supplier A Supplier B 1pg 10 pg NTC 1 h 1pg 10 pg NTC 1.5 h 1pg 10 pg NTC 2 h 1pg 10 pg NTC Fresh 1pg 10 pg NTC 1 h 1pg 10 pg NTC 1.5 h 1pg 10 pg NTC 2 h 1pg 10 pg NTC Fresh 1pg 10 pg NTC 1 h 1pg 10 pg NTC 1.5 h 1pg 10 pg NTC 2 h 1pg 10 pg NTC Figure 5. Superior stability after reaction setup at room temperature. HeLa total RNA (10 and 1 pg) was used as a template for amplification of ACTB in triplicates, according to suppliers’ instructions. Reactions were either set up on ice or left at room temperature for the times indicated before analysis on a 2% agarose gel. Distinct, gene-specific bands are observed with the QIAGEN OneStep Ahead RT-PCR Kit even after a 2-hour incubation at room temperature before cycling (blue arrow), whereas reactions performed with kits from other suppliers deteriorate as time progresses. Gene-specific bands appear significantly weaker, if present at all, while primer–dimers (red arrows) become more prominent.

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