Kapa taq hotstart dna polymerase

DNA was prepared from mouse brain tissues and tails at the age indicated using the MasterPure Complete DNA and RNA Purification Kit (Epicentre Biotechnologies). Samples were incubated with Proteinase K, RNase A treated, followed by protein precipitation and centrifugation to remove cellular debris. DNA was precipitated with isopropanol, washed and resuspended in H2O. Amplification of CAG repeats from Hdh(Q150/wt)/ogg1(+/+), Hdh(Q150/Q150)/ogg1(+/+), Hdh(Q150/wt)/ogg1(-/-), and Hdh(Q150/Q150)/ogg1(-/-) mouse DNA was performed with a HEX-labeled forward primer (CCCATTCATTGCCTTGCTG) and reverse primer (GCGGCTGAGGGGGTTGA) in 15 μl reactions containing 0.2 mM dNTPs, 2 M betaine, AM buffer (67 mM Tris·HCl, pH 8.8/ 16.6 mM (NH4)SO4/ 2 mM MgCl2/ 0.17 mg/ml BSA) and 1 unit of Kapa Taq HotStart DNA Polymerase (Kapa Biosystems). Cycling conditions were as follows; 5 min at 94°C; 30 s at 94°C, 30 s at 60°C, 3 min at 72oC for 5 cycles; 30 s at 94°C, 30 s at 55°C, 5 min at 72°C for 38 cycles; 5 min at 72°C. Genescan analysis was performed using GeneMapper software v4. The statistical program R was used to separate partially overlapping curves in homozygous animals.

Heterozygosity status was assessed through the analysis of 16 microsatellites mapping on chromosomes 17 and 13 (panels 23, 24, and 19 respectively; Thermo Fisher Scientific, Waltham, MA, USA). For chromosome 17 we selected 11 markers: D17S849, D17S831, D17S938, D17S1852, D17S799, D17S798, D17S1868, D17S949, D17S785, D17S784, D17S928; and five markers for chromosome 13: D13S171, D13S153, D13S265, D13S159, D13S158. Polimeration chain reaction (PCR) was performed using Kapa Taq HotStart DNA Polymerase (KAPA Biosystems, Wilmington, MA, USA) under standard conditions and run on an ABI3730 DNA Analyzer. Data were analyzed using GeneMapper Software (Thermo Fisher Scientific, Waltham, MA, USA).