Nov 12, 2024

Public workspaceIsolating DNA from Wolbachia and Drosophila with the QIAwave DNA Blood and Tissue Kit

  • 1Department of Biological Sciences, Lehigh University
  • Shropshire Lab
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Protocol CitationHelene Hartman, J. Dylan Shropshire 2024. Isolating DNA from Wolbachia and Drosophila with the QIAwave DNA Blood and Tissue Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm9xyol3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 12, 2024
Last Modified: November 12, 2024
Protocol Integer ID: 111960
Keywords: DNA extraction, Drosophila, Wolbachia
Abstract
This DNA extraction protocol is column-based and adapted from the manufacturer’s recommendations for the QIAwave DNA Blood and Tissue Kit. It has been successfully used to isolate high-quality genomic DNA from a range of Drosophila tissues, including testes and ovaries. The protocol is also well-suited for research on maternally transmitted Wolbachia endosymbionts.
Materials
Materials
  • ReagentQIAwave DNA Blood & Tissue KitQiagenCatalog #69556
  • ReagentPure Ethanol, 200 ProofKOPTECCatalog #V1001
  • ReagentCentrifuge tubes, safe-lock, PCR clean, 1.5 mLEppendorfCatalog #022363212
  • ReagentPipette tips, filtered, PCR clean and sterile, 0.1 - 10 uLEppendorfCatalog #0030078519
  • ReagentPipette tips, filtered, PCR clean and sterile, 2 - 100 uLEppendorfCatalog #0030078543
  • Reagent Pipette tips, filtered, PCR clean and sterile, 50 - 1,000 uLEppendorfCatalog #0030078578
  • ReagentHomogenizing beads, 2.8 mm, ceramicVWR InternationalCatalog #10158-554

Equipment
  • Bead-mill homogenizer (Benchmark, IPD9600)
  • Centrifuge (Eppendorf, 5420)
  • Dry-bath incubator (Eppendorf, F2.0 Thermomixer)
  • Freezer (any)
  • Fridge (any)
  • Pipette, 10 μL (Eppendorf, 4861000708)
  • Pipette, 100 μL (Eppendorf, 4861000716)
  • Pipette, 1000 μL (Eppendorf, 4861000732)
  • Vortex mixer (Any)
Protocol materials
ReagentCentrifuge tubes, 1.5 mL, safe-lock, PCR cleanEppendorfCatalog #22363212
In Before starting and 4 steps
ReagentHomogenizing beads, 2.8 mm, ceramicVWR InternationalCatalog #10158-554
In Before starting, Materials
ReagentQIAwave DNA Blood & Tissue KitQiagenCatalog #69556
In Before starting, Materials
ReagentPure Ethanol, 200 ProofKOPTECCatalog #V1001
Materials
ReagentCentrifuge tubes, safe-lock, PCR clean, 1.5 mLEppendorfCatalog #022363212
Materials
ReagentPipette tips, filtered, PCR clean and sterile, 0.1 - 10 uLEppendorfCatalog #0030078519
Materials
ReagentPipette tips, filtered, PCR clean and sterile, 2 - 100 uLEppendorfCatalog #0030078543
Materials
Reagent Pipette tips, filtered, PCR clean and sterile, 50 - 1,000 uLEppendorfCatalog #0030078578
Materials
Safety warnings
This protocol involves the use of various chemicals and reagents that require careful handling and strict adherence to safety guidelines to ensure safe laboratory practices. Please review the Material Safety Data Sheets (MSDS) for each reagent before beginning the protocol and take appropriate precautions. All steps should be performed while wearing gloves, a lab coat, and eye protection.
Before start
  1. Collect samples for DNA extraction in ReagentCentrifuge tubes, 1.5 mL, safe-lock, PCR cleanEppendorfCatalog #22363212 . Include 3 ReagentHomogenizing beads, 2.8 mm, ceramicVWR InternationalCatalog #10158-554 in each tube.
  2. Preheat dry-bath incubator (e.g., Eppendorf, F2.0 Thermomixer) to Temperature56 °C .
  3. Clean workspace with Concentration10 % (v/v) bleach and Concentration70 % (v/v) ethanol.
  4. Unpack ReagentQIAwave DNA Blood & Tissue KitQiagenCatalog #69556 . The kit should contain spin columns, collection tubes, Buffer ATL, Buffer AL, Buffer AW1, Buffer AW2, Buffer AE, and Proteinase K. Create aliquots of each solution, as appropriate.
  5. Make sure ethanol has been added to Buffer AW1 and Buffer AW2.
  6. Check Buffer ATL and Buffer AL for precipitates. Dissolve precipitates at Temperature56 °C .
Lyse Cells
Lyse Cells
2h 5m
2h 5m
Transfer ReagentCentrifuge tubes, 1.5 mL, safe-lock, PCR cleanEppendorfCatalog #22363212 containing sample to a bead-mill homogenizer.
Homogenize at Shaker1500 rpm, Room temperature for Duration00:02:00 .
2m
Create lysis mixture containing the following, per sample in a ReagentCentrifuge tubes, 1.5 mL, safe-lock, PCR cleanEppendorfCatalog #22363212 :
  • Amount180 µL Buffer ATL
  • Amount20 µL Proteinase K
1m
Pipetting
Add Amount200 µL lysis mixture to each sample.
1m
Pipetting
Vortex briefly to mix.
1m
Mix
Incubate at Temperature56 °C for a minimum of Duration02:00:00 in a dry-bath incubator. DurationOvernight incubation is also acceptable.
(Optional) If using a Thermomixer, set to shake at Shaker300 rpm .
Four samples in a Thermomixer set to 56oC and 300 rpm.

2h
Temperature
Capture DNA
Capture DNA
6m
6m
While waiting for the lysis incubation, complete the following:
  • Set up 1 set of spin columns.
  • Set up 1 set of ReagentCentrifuge tubes, 1.5 mL, safe-lock, PCR cleanEppendorfCatalog #22363212 .
  • Premix Amount200 µL Buffer AL and Amount200 µL 200 proof ethanol per sample. Make extra.
3m
Pipetting
Add Amount400 µL of Buffer AL/ethanol mixture to each sample.
Immediately vortex for Duration00:00:10 to mix.
Note
It is important that the samples are immediately mixed after the Buffer AL/ethanol mixture is added. Failing to do so can impact DNA yield.

1m
Pipetting
Mix
Critical
Transfer samples to spin columns in collection tubes.
Spin column within a waste collection tube.

1m
Centrifuge for Duration00:01:00 at Centrifigation16000 rcf, Room temperature to capture the DNA on the column membranes.
1m
Centrifigation
Wash DNA
Wash DNA
10m
10m
Empty collection tubes by pouring into a waste container.
1m
Add Amount500 µL Buffer AW1 to each sample.
1m
Pipetting
Centrifuge for Duration00:01:00 at Centrifigation16000 rcf, Room temperature to wash the membranes.
1m
Centrifigation
Wash
Empty collection tubes by pouring into a waste container.
1m
Add Amount500 µL Buffer AW2 to each sample.
1m
Pipetting
Centrifuge for Duration00:01:00 at Centrifigation16000 rcf, Room temperature to wash the membranes.
1m
Centrifigation
Wash
Transfer the columns to new collection tubes.
1m
Centrifuge for Duration00:03:00 at Centrifigation16000 rcf, Room temperature to dry the membranes.
3m
Centrifigation
Elute DNA
Elute DNA
4m
4m
Transfer the columns to clean ReagentCentrifuge tubes, 1.5 mL, safe-lock, PCR cleanEppendorfCatalog #22363212 .
1m
Add Amount40 µL buffer AE to the membrane of each column.
Note
Take care to apply Buffer AE directly to the membrane. Salts are likely to accumulate in the sample if the buffer is applied to the plastic.

1m
Pipetting
Incubate at TemperatureRoom temperature for Duration00:01:00 .
1m
Incubation
Centrifuge for Duration00:01:00 at Centrifigation16000 rcf, Room temperature to elute DNA from the membrane.
Safety information
As the column prevents complete closure of the centrifuge tube lid, position the lid inward toward the center of the centrifuge. Secure the centrifuge rotor lid properly to prevent tube lid breakage during centrifugation.

1m
Centrifigation
Use immediately or store at:
  • Temperature-80 °C for long-term storage
  • Temperature-20 °C for use within a few weeks
  • Temperature4 °C for use within a few days
Pause