QIAamp DNA 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

Sample Pretreatment and DNA Extraction Procedure
Samples must be pretreated to ensure lysis of the cyst cell wall. Procedures are mostly based either on chemical lysis 17 or mechanical lysis based on the thermal-shock procedure. 9,13,18 These two procedures were compared. Amoeba cysts or trophozoites were suspended in 100 μL of PBS, 100 μL of the detergent solution (Tris-HCl 20 mM and SDS 0.5%), and 10 μL of proteinase K (20 minutes at 56°C, then 10 minutes at 95°C), or in 180 μL of a tissue lysis buffer solution (Qiagen, Hilden, Germany) added to 20 μL of proteinase K, incubated for 10 minutes at 56°C, then thermally shocked (10 minutes at 95°C, 3 minutes at −80°C, 1 minute 95°C) and extracted.
Two different extraction procedures were also tested: one based on a manual procedure (Qiagen QIAamp DNA Mini kit) and another using automatic extraction (BioRobot EZ1 Workstation, with EZ1 DNA Tissue card, Qiagen). DNA was eluted in 50 μL of elution buffer AE (Qiagen). PCR was performed on 12 samples containing one cyst submitted to each different pretreatment; on 20 samples containing one, two, or three cysts; and 12 samples containing one, two, or three vegetative forms of A. polyphaga , all thermally shocked but extracted with two different procedures.

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Manufacturer protocol

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

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