Wizard® Genomic DNA Purification Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	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

The DNA were harvested by scraping in ice-cold Nuclei Lysis Solution (200 µl/dish) from the DNA purification kit (Wizard SV Genomic DNA Purification System, Promega). For DNA extraction we followed the manufacturer’s instructions for the kit. The levels of mtDNA were measured by normalizing the mitochondrial cytochrome b gene (MT-CYB, Rn03296746_s1) to the nuclear heat shock protein 70 (Hspa1a, Rn00583013_s1) gene. All samples were run in triplicate in 25 µl reaction volume containing 50 ng sample DNA, 2x Probe RT-Mastermix (Qiagen, Valencia, CA, USA), with both probes in each well. The MT_CYB probe was labeled with VIC, and the Hspa1a probe was labeled with FAM. Amplification conditions were: one cycle of 95°C for 15.05 min, and 45 cycles of 95°C for 15 s and 60°C for 1 min and gene expression levels were quantified by the ΔΔCt method.

VII.b. RNA quantification
Total RNA was harvested by scraping in ice-cold RNA lysis buffer (200 µL/dish). The SV Total RNA Isolation System kit (Promega, Madison, WI, USA) was used to isolate RNA from neuronal cells following the manufacturer’s instructions. From each sample, 50 pg of total RNA was reverse transcribed and amplified using the Qiagen OneStep Probe RT-PCR Kit (Qiagen). Gene-specific primers targeting rat Drp1 (Dnm1l anti-sense primer/probe, Applied Biosystems: # Rn00586466_m1, VIC) and Beta actin were used (Applied Biosystems: # 4352340E, FAM) with 35 cycles for gene amplification. For the quantification of gene expression levels the ΔΔCt method was used.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing DNA isolation / purification Cells - Primary cells Rat cortical neurons using Wizard® Genomic DNA Purification Kit from Promega.

Manufacturer protocol

Download the product protocol from Promega for Wizard® Genomic DNA Purification Kit below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing DNA isolation / purification Cells - Primary cells Rat cortical neurons using Wizard® Genomic DNA Purification Kit from Promega. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.