Introduction

The Biomeme M1 Sample Prep Cartridge Kit for DNA-HI (High Concentration) is a portable way to extract purified nucleic acids from different types of samples. This kit doesn't need laboratory equipment, refrigeration, electricity, incubation, or alcohol precipitation. Instead, it relies on a filtration-based method where nucleic acids attach to the silica membrane inside Biomeme's M1 Sample Prep Columns. The columns are washed several times, resulting in purified nucleic acids upon elution.

This protocol is used in conjunction with Biomeme’s portable qPCR assays.

Safety

• Follow universal lab safety precautions: wear a lab coat and gloves when working in the lab, no open-toed shoes, no eating and drinking.
• DO NOT add bleach or acidic solutions directly to the buffers contained in Biomeme’s M1 Sample Prep cartridges. Guanidine salts will react to create toxic gases.
• All components of the Biomeme M1 Sample Prep Cartridge Kit for DNA-HI should be stored in a dry place at room temperature (15-30°C).

Reagents

Equipment and Consumables

Protocol

Overview

The DNA-HI extraction cartridges consist of six chambers containing pre-aliquoted reagents. Sample homogenate is added to chamber 1, and then a syringe (with a barb column tip) is pumped sequentially in each chamber, moving from left to right. The numbers on the front label indicate the number of cycles the syringe is pumped up and down within a given chamber.

Quick Protocol

1. Homogenize and lyse samples according to sample type (see detailed protocol below).
2. Add 1 ml of sample homogenate to position 1 of the DNA-HI cartridge.
3. Pump a 1 cc syringe with a Barb Column Tip within each chamber according to the following table, moving from left to right:

4. Transfer the eluted DNA to a 2 ml snap-cap tube.
5. Use the eluted DNA immediately or store at -20°C

Detailed Protocol

Homogenization and Lysis - Water Samples

1. Set up the filter apparatus with a new single-use analytical filter funnel containing a 0.2 um filter.
2. Let the collected sample sit undisturbed for a few minutes to allow sand and debris to settle.
3. Pour the water sample into the filter funnel to the 100 ml mark and use the vacuum pump to draw the water through the filter. Repeat with additional 100 ml volumes until the desired volume is reached. Record the total volume of water filtered.

5. Remove the filter from the vacuum flask and discard the cup.
6. Flip the filter and Whatman paper into your gloved hand or filter funnel top. Discard the Whatman paper and then roll up the filter with the transfer side facing inward.
7. Transfer the rolled filter into a Biomeme homogenization tube.
8. Transfer 2 ml of homogenization buffer to the homogenization tube.
9. Recap the homogenization tube and shake vigorously for at least 1 minute to lyse the sample properly.

Homogenization and Lysis - Tick Samples

1. Add the tick(s) to the homogenization tube.
2. Transfer 2 ml of homogenization buffer to the homogenization tube.
3. Recap the homogenization tube and shake vigorously for at least 1 minute to lyse the sample properly.

Extract Samples

1. Attach a 1 cc syringe to a Barb Column Tip (containing a silica membrane), and use to puncture two holes in Position 1 (Red) of the cartridge.
2. Set this syringe with the attached filter aside. You're going to come back to it later.
3. Transfer 1 ml of the sample homogenate (generated above) to Position 1 (Red) with a P1000 and filter tip.
4. Using the syringe set aside in step 2, insert the end of the Barb Column Tip into one of the holes in the cartridge at Position 1 (Red) and slowly pump up and down 10 times.

5. Use the Barb Column Tip to puncture two holes in Position 2 (Red Orange) of the cartridge.
6. Insert the end of the Barb Column Tip into one of the holes in Position 2 (Red Orange) and slowly pump up and down 2 times.
7. Use the Barb Column Tip to puncture two holes in Position 3 (Orange) of the cartridge.
8. Insert the end of the Barb Column Tip into one of the holes in Position 3 (Orange) and slowly pump up and down 1 time.
9. Use the Barb Column Tip to puncture two holes in Position 4 (Yellow) of the cartridge.
10. Insert the end of the Barb Column Tip into one of the holes in Position 4 (Yellow) and slowly pump up and down 1 time.
11. Use the Barb Column Tip to puncture one hole in Position 5 (Gray) of the cartridge.
12. Insert the end of the Barb Column Tip into the hole in Position 5 (Gray) and rapidly pump up and down 20 times, or until the column appears dry.
13. Use the Barb Column Tip to puncture two holes in Position 6 (Green) of the cartridge.
14. Insert the end of the Barb Column Tip into one of the holes in Position 6 (Green) and slowly pump up and down 2 times.
15. Use your syringe and sample tip to transfer the ~1 ml of purified DNA to a snap-cap tube. Label the tube with sample ID, and record the location and other details in your lab notebook and in the Water Metadata and QPCR Results Document.
16. Use the eluted DNA immediately or store at -20°C.

Cleanup

1. Used cartridges can be discarded in the normal trash. Place the used cartridges in a sealed ziplock bag containing paper towels before disposal.
2. Rinse the hand pump with tap water and shake out excess. Allow to air dry.
3. Dispose of filtered water down the drain.
4. Rinse the vacuum flask and tubing 3 times with tap water and then allow to air dry.