QIAamp DNA Micro 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

DNA extraction
In order to determine the minimum amount of samples to obtain sufficient DNA for analysis, we collected 5–25 LM separately cut sections and processed them for H. pylori-DNA isolation. The extraction procedure was performed with QIAamp DNA Micro Kit (Quiagen) with the following modifications:

1.
30 μL of tissue lysis buffer (ATL) and 20 μL proteinase K were added to each laser micro-dissected sample collected in a 0.2 mL tube.

2.
The samples collected were mixed in a vortex for 15 s and then placed in a thermo-block with the lid down because the sections of tissue were attached to the top (Graphical abstract). Samples were incubated for 6 h at 56 °C with occasional vortexing pulses. Then, 50 μL of ATL were added. Each sample was mixed and the lid reviewed with a magnifying glass to confirm that all sample sections had been lysed.

3.
The complete material was transferred to a new 1.5 mL tube. Then 100 μL of working AL solution (2 μL of CAR was mixed with 98 μL Buffer AL) was added and mixed in a vortex for 15 s.

4.
Then, 100 μL of molecular-grade ethanol was added and mixed in a vortex for 15 s. The sample was incubated for 5 min at room temperature.

5.
The sample was then centrifuged in the spin mode for 10 s to remove drops from inside the lid.

6.
Approximately 300 μL of the lysate sample was transferred to the QIAamp MinElute column placed into a 2 mL collection tube. The sample was centrifuged at 8000 rpm for 1 min and the column was placed in a new tube.

7.
The column was then washed with 700 μL Buffer AW1 and centrifuged at 8000 rpm for 1 min. The column was once again placed in a new collection tube.

8.
Then 700 μL of AW2 buffer was added to the column and the centrifugation protocol was repeated to discard the AW2 buffer.

9.
The QIAamp MinElute column, adapted to a new 2 mL collection tube, was centrifuged at 14,000 rpm for 3 min to dry the membrane.

10.
The last collection tube was replaced with a 1.5 mL tube (previously labeled with sample identification number).

11.
Then 35 μL of the AE Buffer was dispensed in the center of the column. After 5 min incubation at 20–25 °C, the sample was centrifuged at 14,000 rpm for 1 min.

12.
The isolated DNA in the 1.5 mL tube was stored at −20 °C until assayed. The DNA was quantified with Picogreen, (INVITROGEN™) following standard instructions recommended by the manufacturer.

13.
To isolate DNA from the conventional FFPE, five tape sections were cut with a new disposable blade in a semi-automatic microtome (Shandon Finesse Me+, Thermo Scientific) and were directly collected in a 1.5 mL sterile Eppendorf tube. Then DNA extraction was performed according to suggested manufacturer instructions (QIAamp DNA Micro Kit Quiagen).

14.
Data showed that the greatest number of tissue sections obtained with LM yielded greatest concentrations of DNA. The average DNA concentration obtained from 25 LM cut sections was 1.94 ± 0.16 ng/μL. The DNA concentration obtained from LM samples was 10-fold less than the concentration obtained following conventional extraction from FFPE gastric biopsies (Fig. 4). Fig. 5 indicates the consistency of the method to isolate microbial DNA. Although the final concentration of DNA isolated by this method is less than the amount of DNA isolated by conventional strategies, the obtained nucleic acid is of bacterial origin.

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Manufacturer protocol

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

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