Gc rich pcr system amplification buffer

For both samples, genomic DNA was extracted using a high-capacity membrane-based column (QuickGene810, AutoGen, Inc., Holliston, MA) and was quantitated using an A260/A280 ratio with a NanoDrop spectrophotometer (ThermoScientific, Inc., Wilmington, DE) and agarose gel electrophoresis. The DRD4 VNTR polymorphism was amplified, with 0.2 μM of DRD4 forward primer 5′-GCGACTACGTGGTCTACTCG and 0.2 μM of DRD4 reverse primer 5′-AGGACCCTCATGGCCTTG [47 (link)], using the Roche GC-Rich PCR System amplification buffer (Roche Applied Science, Inc., Mannheim, Germany) and 20 ng of genomic DNA in a volume of 25 μl. A Stratagene thermocycler (Life Technologies, Inc., Grand Island, NY) was used to heat the samples at 95 °C for 3 min, then cycled 40 times at 95 °C for 20 s, 57 °C for 20 s, and 72 °C for 1 min, followed by 72 °C for 3 min. Polymerase chain reaction products were separated and visualized on a 2% agarose gel (type 1-A, Sigma, St. Louis, MO) stained with ethidium bromide (Lichter, Barr et al. 1993). DNA genotyping was performed blind to the children’s behaviour and phenotype.

Oragene Saliva kit OG-500 was used for saliva collection (DNA Genotek, Ottawa, ON, Canada). Genomic DNA was extracted using a high-capacity membrane-based column (QuickGene810, AutoGen, Inc., Holliston, MA, USA), and was quantitated using A260/A280 ratio (Nanodrop), and agarose gel electrophoresis. The DRD4 variable number tandem repeat (VNTR) polymorphism was amplified using 0.2 µM of each primer DRD4 forward 5′-GCGACTACGTGGTCTACTCG and DRD4 reverse 5′-AGGACCCTCATGGCCTTG [17 (link)], using the Roche “GC-Rich PCR System” amplification buffer (Roche Applied Science, Inc., Mannheim, Germany) and 20 ng of genomic DNA in a volume of 25 µL. The samples were heated in a Stratagene thermocycler (Life Technologies, Inc., Grand Island, NY, USA) at 95 °C for 3 min, then cycled 40 times at 95 °C for 20 s, 57 °C for 20 s, and 72 °C for 1 min, followed by 72 °C for 3 min. PCR products were separated and visualized on a 2% agarose gel (type 1-A, Sigma, St. Louis, MO, USA) stained with ethidium bromide.