Wizard® Genomic DNA Purification Kit

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	For blood DNA isolation, blood must be collected in EDTA, heparin or citrate anticoagulant tubes to prevent clotting.

Protocol tips
For blood DNA isolation, blood must be collected in EDTA, heparin or citrate anticoagulant tubes to prevent clotting.

Publication protocol

2.12 Platinum uptake and incorporation into DNA
Uptake and incorporation of platinum was measured by atomic absorption or by immunochemical detection with a specific antibody. For cisplatin uptake, the cells were incubated with different amounts of cisplatin for the indicated times, after which ~106 cells were suspended in 200 µl PBS and 400 µl HNO3 and placed in an ultrasonic bath for 5 min. Platinum was measured by flameless atomic absorption on an PerkinElmer 4100ZL atomic absorption spectrometer. To measure cisplatin uptake into mitochondria, cells were lysed and mitochondria were isolated as described above. Mitochondria were dissolved in 1.5 ml of 5 M nitric acid and incubated for 24 h at room temperature. Platinum was quantitated by inductively coupled plasma mass spectrometry. For measurement of platinum adducts in DNA, the cells were incubated with cisplatin for 5 and 16 h. Genomic DNA was extracted from the cells using the Wizard Genomic DNA Isolation kit as directed by the supplier. For mitochondrial DNA isolation, mitochondria were first prepared using the Mitochondria Isolation Kit described above and DNA was prepared from the mitochondria using the Wizard kit mentioned above. Equal aliquots of DNA were spotted onto nitrocellulose membranes and cross-linked by UV irradiation. Diguanosine-cisplatin adducts were detected immunochemically with a monoclonal antibody and HRP-conjugated secondary antibodies (Abcam).

2.13 Measurement of mitochondrial DNA
The mitochondrial DNA copy number was measured according to the procedure used by Dan et al (2015). HeLa cells were transfected with 10 nM HCLPP-siRNA for 5 hours, cells were transferred to fresh DMEM medium and grown for 16 h, after which they were treated with 2.5 µg/ml cisplatin for 0 or 24 h. The cells were harvested and total DNA was isolated using a Wizard Genomic DNA Purification Kit (Promega, San Luis Obispo, CA). The mitochondrial CYTB and the nuclear β-actin gene copy numbers were measured by quantitative PCR using the SYBR Green CFX96 Real-Time System (Bio-Rad Laboratories, Hercules, CA). Each reaction contained 100 ng DNA for CYTB and 250 ng DNA for actin. PCR amplification was performed under the following condition: denaturation at 95 C for 10 min, followed by 40 two-step cycles (95 C for 15 s and 60 C for 1min). The primers for CYTB were: upper 5’-ACTATCCGCCATCCCATAC-3’; Lower 5’-GCAAGAATAGGAGGTGGAG-3’. The primers for β-actin were: Upper 5’-ACCTTCTACAATGAGCTGCG-3’; Lower 5’-CCTGGATAGCAACGTACATGG-3’. Three independent cultures of each cell line were grown and duplicate DNA samples from each culture were analyzed. For each sample delta delta Cq measurements were used to obtain the ratio of the CYTB and ACTB copy numbers in the reaction. The average CYTB copy numbers per cell were calculated to obtain mitochondrial DNA copy number. Standard among the six samples from each cell line were <10% of the measured values; P was <0.01 for calculated values of the cellular copy numbers.



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