Nov 13, 2024
Isolating DNA from Wolbachia and Drosophila with the Puregene Tissue Kit
- 1Department of Biological Sciences, Lehigh University
- Shropshire Lab
Protocol Citation: Alphaxand Njogu, J. Dylan Shropshire 2024. Isolating DNA from Wolbachia and Drosophila with the Puregene Tissue Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn987ml5d/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 10, 2024
Last Modified: November 13, 2024
Protocol Integer ID: 111851
Keywords: DNA extraction, Drosophila, Wolbachia
Abstract
This protocol is a modified version of the Puregene Tissue Kit manufacturer's protocol. It has been successfully used to isolate high-quality genomic DNA from various Drosophila tissues, including testes and ovaries. This method has also been used to extract DNA from the maternally transmitted Wolbachia endosymbiont of Drosophila.
Materials
Materials
- Puregene Tissue Kit (4 g)QiagenCatalog #158063
- IsopropanolFisher ScientificCatalog #BP2618500
- 70 % (v/v) ethanol from Pure Ethanol, 200 ProofKOPTECCatalog #V1001 and molecular-grade H2O
- Centrifuge tubes, safe-lock, PCR clean, 1.5 mLEppendorfCatalog #022363212
- Pipette tips, filtered, PCR clean and sterile, 0.1 - 10 uLEppendorfCatalog #0030078519
- Pipette tips, filtered, PCR clean and sterile, 2 - 100 uLEppendorfCatalog #0030078543
- Pipette tips, filtered, PCR clean and sterile, 50 - 1,000 uLEppendorfCatalog #0030078578
- Glycogen, UltraPureInvitrogen - Thermo FisherCatalog #10814010
- Homogenizing 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)
- Vacuum centrifuge (Eppendorf, Vacufuge Plus)
Protocol materials
Glycogen, molecular biology gradeInvitrogenCatalog #10814-010
Step 10
Pipette tips, filtered, PCR clean and sterile, 0.1 - 10 uLEppendorfCatalog #0030078519
Materials
Puregene Tissue Kit (4 g)QiagenCatalog #158063
Materials
Pure Ethanol, 200 ProofKOPTECCatalog #V1001
Materials
Homogenizing beads, 2.8 mm, ceramicVWR InternationalCatalog #10158-554
In Materials, Before starting
Puregene Tissue KitQiagenCatalog #158063
Before starting
Isopropanol, molecular biology gradeFisher ScientificCatalog #BP-2618-1
Step 9
IsopropanolFisher ScientificCatalog #BP2618500
Materials
Pipette tips, filtered, PCR clean and sterile, 2 - 100 uLEppendorfCatalog #0030078543
Materials
Glycogen, UltraPureInvitrogen - Thermo FisherCatalog #10814010
Materials
Centrifuge tubes, safe-lock, PCR clean, 1.5 mLEppendorfCatalog #022363212
Materials
Pipette tips, filtered, PCR clean and sterile, 50 - 1,000 uLEppendorfCatalog #0030078578
Materials
Centrifuge tubes, 1.5 mL, safe-lock, PCR cleanEppendorfCatalog #22363212
In Before starting and 3 steps
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
- Collect samples for DNA extraction in Centrifuge tubes, 1.5 mL, safe-lock, PCR cleanEppendorfCatalog #22363212 . Include 3 Homogenizing beads, 2.8 mm, ceramicVWR InternationalCatalog #10158-554 in each tube.
- Preheat dry-bath incubator (e.g., Eppendorf, F2.0 Thermomixer) to 55 °C .
- Clean workspace with 10 % (v/v) bleach and 70 % (v/v) ethanol.
- Unpack Puregene Tissue KitQiagenCatalog #158063 . The kit should contain cell lysis solution, protein precipitation solution, DNA hydration solution, and Proteinase K. Create aliquots of each solution, as appropriate.
Lysis
Lysis
3h 6m
3h 6m
Transfer Centrifuge tubes, 1.5 mL, safe-lock, PCR cleanEppendorfCatalog #22363212 containing sample to a bead-mill homogenizer.
Homogenize at 1500 rpm, Room temperature for 00:02:00 .
2m
Create lysis mixture containing the following, per sample in a Centrifuge tubes, 1.5 mL, safe-lock, PCR cleanEppendorfCatalog #22363212 :
- 300 µL cell lysis solution
- 1.5 µL Proteinase K
Prepare a bit extra to account for pipetting errors.
1m
Add 301.5 µL lysis mixture to each sample.
2m
Mix by inverting the tubes 25 times.
Note
To process large batches, arrange tubes in a tube rack. Place a second rack on top, sandwiching the tubes. Invert the entire stack to simultaneously invert all tubes.
1m
Incubate at 55 °C for between 03:00:00 and 16:00:00 in a dry-bath incubator.
(Optional) If using a Thermomixer, set to shake at 300 rpm .
3h
Protein precipitation
Protein precipitation
5m
5m
Transfer samples to an ice block.
Incubate for 00:01:00 to quickly cool the samples.
1m
Add 100 µL protein precipitation solution to each sample.
Immediately vortex each sample for 00:00:20 .
Note
It is important that the samples are immediately mixed after the protein precipitation solution is added. Failing to do so can impact quality of the precipitation.
1m
Centrifuge for 00:03:00 at 16000 rpm, Room temperature to pellet the protein.
If the protein pellet is not tight, incubate On ice for 00:05:00 and repeat the centrifugation.
3m
DNA precipitation
DNA precipitation
4m 15s
4m 15s
Add 300 µL Isopropanol, molecular biology gradeFisher ScientificCatalog #BP-2618-1 to a clean Centrifuge tubes, 1.5 mL, safe-lock, PCR cleanEppendorfCatalog #22363212 .
Carefully transfer the supernatant from the prior step to the isopropanol by pipetting.
1m
(Optional) Dilute 20 µg/µL Glycogen, molecular biology gradeInvitrogenCatalog #10814-010 to 0.5 µg/µL . Add 1 µL to each sample.
Note
Glycogen acts as a carrier molecule, improving DNA precipitation yield, particularly in low-biomass samples.
30s
Mix by inverting the tubes 50 times.
1m
Centrifuge for 00:01:00 at 16000 rcf, Room temperature to pellet the DNA.
1m
Carefully discard the supernatant by pouring into a waste container.
Drain the tube by inverting on a paper towel.
Take care that the pellet remains in the tube.
Note
A single, rapid inversion is essential. Allowing the liquid to settle back to the bottom can dislodge the DNA pellet, leading to loss of yield.
45s
Wash with ethanol
Wash with ethanol
16m
16m
Add 300 µL of 70 % (v/v) ethanol to each sample.
30s
Mix by inverting the tubes 50 times.
1m
Centrifuge for 00:01:00 at 16000 rcf, Room temperature to pellet the DNA.
1m
Carefully discard the supernatant by pouring into a waste container.
30s
Washing the pellet once more: go to step #14 .
3m
Transfer samples to a vacuum centrifuge.
Dry the DNA pellets for 00:10:00 . If using a Vacufuge plus, use the D-AL setting.
Note
Overdrying the DNA pellet can significantly reduce its solubility. It is important to stop the drying process before the pellet becomes completely dry.
10m
DNA hydration
DNA hydration
1h 0m 35s
1h 0m 35s
Add between 15 µL and 100 µL DNA hydration solution to each sample.
Note
The choice of elution volume depends on the starting material.
For low biomass samples, a lower volume is preferred.
30s
Vortex for 00:00:05 to break up the DNA pellet.
5s
Choose 1:
- Incubate at 65 °C for 01:00:00 to dissolve the DNA.
- Incubate at Room temperature Overnight to dissolve the DNA.
1h
Use immediately or store at:
- -80 °C for long-term storage
- -20 °C for use within a few weeks
- 4 °C for use within a few days