AllPrep DNA/RNA Mini 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

Colonic mucosal biopsies, 2–3 mm in diameter, were collected from two patients undergoing planned colectomy at Akershus University Hospital, and from three patients undergoing colonoscopy as part of the EU-project IBD-Character (EU ref no 305676). Prior to the colectomy and colonoscopy the patients performed a bowel cleansing by Picoprep (Ferring Legemidler AS, Oslo, Norway) according to manufacturer’s instructions. All biopsies were immediately placed in Allprotect Tissue Reagent (Qiagen, Hilden, Germany) and stored according to manufacturer’s instructions. For each of the different protocols tested, one to two biopsies from each patient were used due to a limited number of biopsies (Table 1).

Total RNA and DNA were purified from colonic mucosal tissue using the AllPrep DNA/RNA Mini Kit (Qiagen). Manufacturer’s instructions were followed with the exception of the lysis steps, where three tissue lysis protocols (Protocol 1, 2 and 3) were performed and evaluated (Fig. 1; Additional file 1). For RNA purification, DNase treatment was included and performed on column as described in the AllPrep DNA/RNA Mini Kit protocol. RNA and DNA were eluted using 40 µl nuclease free water (NFW) and stored at −80 and −20 °C, respectively.

The resulting RNA quantity and quality data from the tested protocols were compared to RNA quantity and quality data obtained using a standard RNA purification protocol combining Qiazol, phase separation and kit column based purification [16] (Table 1; Additional file 1). The resulting DNA quantity and quality data were compared to DNA quantity and quality data from a combination of mechanical and enzymatic pre-treatments as recommended by Qiagen for lysis of Gram-positive bacteria, followed by purificaton with AllPrep DNA/RNA Mini Kit (kit 1), the QIAamp DNA Stool Mini Kit (kit 2) and the DNeasy Blood & Tissue Kit (kit 3) (Table 1; Additional file 1). For all three purification kits the manufactures instructions were followed after pre-treatment. DNA was eluted using 40 µl NFW and stored at −20 °C.

The concentration of the RNA and DNA samples were assessed using NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). For RNA quality the RNA integrity number (RIN) was tested using the Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA, USA) and the 2100 Expert Software. The assay and reagent kit used were Eukaryote Total RNA Nano Series II and the Agilent RNA 6000 Nano Kit. For DNA the quality was obtained using the ratio 260/280 for assessing the purity of the samples. The molecular size of the genomic DNA was measured in a 1 % agarose gel in 1xTBE, 70 V, running time 2.5 h (Fig. 2).

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Manufacturer protocol

Download the product protocol from Qiagen for AllPrep DNA/RNA Mini Kit below.

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