Jun 08, 2022
Version 4
DNA Extract wash from filter paper stored at room temperature V.4
- Katie Izenour1,
- Anwar Kalalah1,
- Fayez Salib2,
- Sarah Zohdy1
- 1Auburn University College of Veterinary Medicine;
- 2Cairo University Faculty of Veterinary Medicine
Protocol Citation: Katie Izenour, Anwar Kalalah, Fayez Salib, Sarah Zohdy 2022. DNA Extract wash from filter paper stored at room temperature. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxp2nzl8j/v4Version created by Katie Izenour
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: June 08, 2022
Last Modified: June 08, 2022
Protocol Integer ID: 64163
Keywords: DNA Extraction, Egypt, Field work, Tropbio filter paper, room temperature DNA storage
Abstract
Preservation of samples requiring cold-chain storage is an often unavoidable challenge especially when doing laboratory work outside the western world. Samples are a precious commodity and it is imperative to maintain their viability. One method for collection and storage of samples in a liquid form (blood, saliva, serum) at room temperature is on filter paper cards like the Cellabs Tropbio Filter Paper Blood Collection Disks™. Methods and protocols exist for the use and recovery of whole blood from filter paper discs. This protocol describes the wash and recovery of DNA extracted from whole blood using the Qiagen DNeasy Extraction kit and dried on filter paper, re-suspended after several months of room temperature storage and use in conventional PCR.
This protocol was used to suspend DNA extracted from the wholeblood of Dogs in Cairo, Egypt. The DNA was extracted using the Qiagen DNEasy Extraction Kit and placed on Cellabs Tropbio Filter Paper Blood Collection Disks™ (filter paper). The DNA dried on the filter paper overnight and was stored in plastic self-closure baggies for several months before being washed off and used in Conventional PCR. The use case described here is of a dog who was PCR positive for Babesia spp. using a sample of whole blood washed from the filter paper as well as dried DNA from filter paper.
Materials
QIAgen DNeasy Blood and Tissue Kit, 50 rxnQiagenCatalog #69504
Tropbio Filter Paper Blood Collection Disks™Catalog #FP
Taq PCR Master Mix KitQiagenCatalog #201445
Tris baseSigma AldrichCatalog #T1503
Gel Red Nucleic Acid Gel StainBiotiumCatalog ##41003
1kb ladderBiotiumCatalog #99962
General:
Pipettes, tips, hood, tabletop centrifuge, microcentrifuge tubes, rocking platform, thermal cycler, gel electrophoresis rig, UV light box, gloves, sterile 1L glass bottle, balance, autoclave, pH meter, distilled water
Protocol materials
QIAgen DNeasy Blood and Tissue Kit, 50 rxnQiagenCatalog #69504
Tropbio Filter Paper Blood Collection Disks™Catalog #FP
Taq PCR Master Mix KitQiagenCatalog #201445
Tris baseMerck MilliporeSigma (Sigma-Aldrich)Catalog #T1503
Gel Red Nucleic Acid Gel StainBiotiumCatalog ##41003
1kb ladderBiotiumCatalog #99962
QIAgen DNeasy Blood and Tissue Kit, 50 rxnQiagenCatalog #69504
Tris baseMerck MilliporeSigma (Sigma-Aldrich)Catalog #T1503
Taq PCR Master Mix KitQiagenCatalog #201445
Gel Red Nucleic Acid Gel StainBiotiumCatalog ##41003
1kb ladderBiotiumCatalog #99962
Whole blood collection from animal
Whole blood collection from animal
Using a sterile syringe and needle, collect 1-5 mL of whole blood from animal's forelimb. Immediately express the contents of the syringe into a new, clean EDTA tube and invert several times to mix.
Whole blood storage
Whole blood storage
IF EXTRACTING DNA ON THE SAME DAY AS BLOOD COLLECTION - Place EDTA tube containing fresh blood on ice or at 4 °C until ready to extract.
IF EXTRACTING DNA AT A LATER DATE FROM BLOOD COLLECTION- Place EDTA tube containing fresh blood in the freezer at -20 °C until ready to extract; sample will need to be thawed and brought to room temperature before DNA extraction can begin.
DNA Extraction
DNA Extraction
If using blood frozen in EDTA, bring it fully to room temperature before beginning to extract
Set up a clean workspace in a biosafety cabinet or other appropriate laboratory space for working with biological material and DNA
Use the QIAgen DNeasy Blood and Tissue Kit, 50 rxnQiagenCatalog #69504
Recommend using the maximum amount of time used for each step, modifications as follows:
1) Protocol Step 4 states: Place a 1.5 mL microcentrifuge tube in a thermomixer or heated orbital incubator with shaking at 900 rpm, 56°C for 01:00:00
Instead: Place the 1.5 mL tube in a thermomixer or heated orbital incubator and incubate at 900 rpm, 56°C for 02:00:00
2) Protocol Step 17 states: Apply 20-100 µL Buffer ATE or distilled water to the center of the membrane.
Instead: Apply 100 µL of Buffer AE to wash DNA into microcentrifuge tube
3) Protocol Step 18: Close the lid and incubate at room temperature for 1 minute. Centrifuge at full speed (20,000 xg; 14,000 rpm) for 1 minute.
Instead: Close the lid and incubate at room temperature for 5 minute. Centrifuge at full speed (20,000 xg; 14,000 rpm) for 1 minute.
3h
The DNA is recovered at the final step of the extraction process when buffer 'AE' is used. Take care not to accidentally discard the flow-thru of this step, the AE wash product contains the actual DNA
Prepare Filter Paper
Prepare Filter Paper
5m
5m
Wear clean gloves and remove individual filter paper disk with 'ears' (each Cellabs Tropbio Filter Paper Blood Collection Disks™- Disk has 6 individual ears attached to it) from the sheet.
Filter paper will need a clean, dry, flat area to dry Overnight after application of the DNA extract, ensure an appropriate bench space is prepared before removing Filter Paper from packaging.
5m
use a sharpie or other permanent marker to write your sample ID on the center part of the disk
if extract is not already at room temperature, bring the DNA extract to room temperature
Apply DNA to Filter Paper
Apply DNA to Filter Paper
5s
5s
Vortex or invert microcentrifuge tube containing DNA extract for 00:00:05 to mix DNA evenly.
On each 'ear' of the filter paper disk, pipette 6 µL of DNA extract
5s
Allow Filter Paper to dry Overnight , or longer on a clean, dry, flat surface
The remaining DNA in the microcentrifuge tube can be discarded or stored in a freezer.
5s
Filter Paper Storage
Filter Paper Storage
After drying overnight, dried filter paper can be placed individually in self-closing plastic bags and sealed tightly. The individual plastic bags can be stored in a larger self-closing plastic bag containing a pelleted desiccant to minimize the risk of moisture getting to the filter paper.
Dried filterpaper cards sealed in individual plastic bags
Dried filter paper stored in individual plastic bags with silica
DNA on filter paper can be stored at room temperature for up to 6 months
Prepare to wash DNA from Filter Paper - make Tris-HCL
Prepare to wash DNA from Filter Paper - make Tris-HCL
3h
3h
10mM Tris-HCL, pH 8.5 is used to wash dried DNA from filter paper. Tris baseSigma AldrichCatalog #T1503
To make 500 mL 10mM Tris-HCL, pH 8.5 wash do the following:
3h
to make 500mL 10mM Tris-HCL, pH 8.5 (Starting with 121.1 g/mol tris powder)
measure 0.6055 g tris base powder and pour into a sterile glass bottle
add about 300mL DI water to the bottle with the tris base powder
Use a pH meter to adjust the pH of the tris base in water until a pH of 8.5 is achieved.
Once the appropriate pH is achieved, add DI water to bring total volume to 500mL
Loosely place a cap on the glass bottle containing the tris base and autoclave it on settings appropriate for your machine
Allow tris base to cool to room temperature following completion of autoclave
Wash Extracted DNA from filter paper
Wash Extracted DNA from filter paper
In a clean, sterile 2mL microcentrifuge tube, place one whole filter paper 'ear' containing dried DNA extract.
Pipette 150µL of sterile Tris HCL @pH 8.5 (made in step 7 above) into the microcentrifuge tube containing the filter paper 'ear'
ensure the filter paper ear is completely submerged in the liquid, close lid, vortex thoroughly for 20 seconds
Ensure the filter paper ear is submerged following vortex, with lid closed, place tube on rocking platform at 4C, 500rpm for 2 hours
following 2 hours of rocking, the DNA will be suspended in the eluate in the tube.
Polymerase Chain Reaction
Polymerase Chain Reaction
Prepare a clean and sterile bench space for PCR
Prepare mastermix following the protocol for the QiagenTaq PCR Master Mix Kit (Catalog #201445). Taq PCR Master Mix KitQiagenCatalog #201445
MasterMix:
| A | B | |
| Qiagen MasterMix | 50 µL | |
| Forward Primer | 3µL | |
| Reverse Primer | 3µL | |
| Sample (washed DNA from step 8) | 10µL | |
| Water | 34µL | |
| Total | 100 µL |
thaw (if necessary) and vortex the microcentrifuge tube with the washed DNA Sample before adding it to the mastermix
use 10µL of the washed DNA (from step 9 of this protocol) as the 'sample' in the mastermix
Thermal Cycle
Thermal Cycle
Thermal Cycle
lid 105°C, 95°C 3min, 95°C 30sec, 60°C 30 sec 72°C 45sec, repeat 34x, 72°C 10min, 4°C infinite hold or placed in 4°C refrigerator until gel electrophoresis
Gel Electrophoresis
Gel Electrophoresis
2% Agarose Gel Electrophoresis with Biotium Gel RedGel Red Nucleic Acid Gel StainBiotiumCatalog ##41003 and 1kb ladder 1kb ladderBiotiumCatalog #99962