QIAamp DNA Blood Midi Kit

DNA isolation / purification Tissue - blood / plasma

Experiment
DNA isolation / purification Tissue - blood / plasma
Product
QIAamp DNA Blood Midi Kit from Qiagen
Manufacturer
Qiagen

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

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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Discussion

Discussion

5 years ago

Author: Denmark

What DNA isolation kit would work for insect samples?

Hello everyone! I am currently using different DNA isolation kits to extract DNA from insects. Even though I am able to successfully extract DNA I would like to maximize the yield. Do you have any tips that might help me with that even if the kits are not specifically designed for insect samples?

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Papers

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Manufacturer protocol

Download the product protocol from Qiagen for QIAamp DNA Blood Midi Kit below.

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Videos

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