QIAamp DNA Blood Midi Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
		- Include RNAse treatment for 15-20 min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C - Use prewarmed TE buffer to elute the DNA

Downstream tips
- Include RNAse treatment for 15-20 min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C - Use prewarmed TE buffer to elute the DNA

Publication protocol

Pharmacogenetic analysis. Genomic DNA was extracted from 1 mL of whole blood (n = 5) or plasma (n = 92) using the Gentra PureGene Blood Kit (Gentra, Minneapolis, MN) and the QIAamp DNA Blood midi kit (Qiagen, Inc., Valencia, CA), respectively, following the manufacturer's instructions, and was reconstituted in a buffer containing 10 mmol/L Tris (pH 7.6) and 1 mmol/L EDTA. SNPs in CYP2C8 were identified from the literature (22, 23). Variations in the genes of interest [CYP2C8*2 (805A>T, I269F), CYP2C8*3 (416G>A, R139K), CYP2C8*4 (792C>G, I264M), CYP3A4*3 (1334T>C, M445T), CYP3A5*3C (6986A>G, splice variant), and ABCB1 3435C>T (I1145I)] were analyzed using Pyrosequencing. Analysis of the CYP3A4, CYP3A5, and ABCB1 genes was done as previously described (24). PCR primers were designed using Primer Express version 1.5 (ABI, Foster City, CA) for CYP2C8*2 (forward, 5′-biotin-CCATGATGTTTAGTGCAGGCC-3′; reverse, 5′-CTCTACTCATTGATTGTTTCCCAGG-3′), CYP2C8*3 (forward, 5′-biotin-ACGCAGAGTAGAGTCACCCACC-3′; reverse, 5′-ATTCTCCCAGTTTCTGCCCC-3′), and CYP2C8*4 (forward, 5′-TGTCAAGCATTACTGGCCTG-3′; reverse, 5′-biotin-CTCTACTCATTGATTGTTTCCCAGG-3′). The Pyrosequencing primers were designed using the Pyrosequencing SNP Primer Design Version 1.01 software (http://www.pyrosequencing.com). CYP2C8 Pyrosequencing primer sequences were as follows: CYP2C8*2 reverse, 5′-TTATCGATTGCTTCCTG-3′; CYP2C8*3 reverse, 5′-TGGGATGGGGAAGA-3′; and CYP2C8*4 forward, 5′-TTGATCAGGAAGCAATC-3′. PCR was carried out using Amplitaq Gold PCR master mix (ABI), 5 pmol of each primer, and 5 to 10 ng of DNA isolated from whole blood or DNA from 1 μL of undiluted plasma. Pyrosequencing was carried out using the Pyrosequencing hsAPSQ96 instrument and software (Biotage, Uppsala, Sweden). The genotype was called variant if it differed from the Refseq consensus sequence for the SNP position (http://www.ncbi.nlm.nih.gov/RefSeq/). Genotype-frequency analysis of Hardy-Weinberg equilibrium was carried out using Clump version 1.9 (25).



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Manufacturer protocol

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