TITLE: DNA Extract was from filter paper stored at room temperature
AUTHORS: Katie  Izenour, Anwar Kalalah, Fayez Salib, Sarah    Zohdy

[DESCRIPTION]
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. 

[STEPS]
SECTION: Whole blood collection from animal
1. 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. 


SECTION: Whole blood storage
2. 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.

SECTION: DNA Extraction
3. 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
SECTION: DNA Extraction
3.1. Use the  


SECTION: DNA Extraction
3.2. 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   for 60 min

Instead:   Place the 1.5 mL tube in a thermomixer or heated orbital incubator and incubate at       for 120 min 


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


4) 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.

SECTION: DNA Extraction
3.3. 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
SECTION: Prepare Filter Paper
4. 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 5 min after application of the DNA extract, ensure an appropriate bench space is prepared before removing Filter Paper from packaging.  

SECTION: Prepare Filter Paper
4.1. use a sharpie or other permanent marker to write your sample ID on the center part of the disk
SECTION: Prepare Filter Paper
4.2. if extract is not already at room temperature, bring the DNA extract to room temperature
SECTION: Apply DNA to Filter Paper
5. Vortex or invert microcentrifuge tube containing DNA extract for 5 s to mix DNA evenly.

On each 'ear' of the filter paper disk, pipette 6 µL of DNA extract

SECTION: Apply DNA to Filter Paper
5.1. Allow Filter Paper to dry 5 s, or longer on a clean, dry, flat surface
The remaining DNA in the microcentrifuge tube can be discarded or stored in a freezer.
SECTION: Filter Paper Storage
6. 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.      

SECTION: Filter Paper Storage
6.1. DNA on filter paper can be stored at room temperature for up to 6 months
SECTION: Prepare to wash DNA from Filter Paper - make Tris-HCL
7. 10mM Tris-HCL, pH 8.5 is used to wash dried DNA from filter paper.

To make 500 mL 10mM Tris-HCL, pH 8.5 wash do the following:
SECTION: Prepare to wash DNA from Filter Paper - make Tris-HCL
7.1. 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
SECTION: Prepare to wash DNA from Filter Paper - make Tris-HCL
7.2. Use a pH meter to adjust the pH of the tris base in water until a pH of 8.5 is achieved.
SECTION: Prepare to wash DNA from Filter Paper - make Tris-HCL
7.3. once the appropriate pH is achieved, add DI water to bring total volume to 500mL
SECTION: Prepare to wash DNA from Filter Paper - make Tris-HCL
7.4. Loosely place a cap on the glass bottle containing the tris base and autoclave it on settings appropriate for your machine
SECTION: Prepare to wash DNA from Filter Paper - make Tris-HCL
7.5. Allow tris base to cool to room temperature following completion of autoclave
SECTION: Wash Extracted DNA from filter paper
8. In a clean, sterile 2mL microcentrifuge tube, place one whole filter paper 'ear' containing dried DNA extract. 
SECTION: Wash Extracted DNA from filter paper
8.1. Pipette 150µL of sterile Tris HCL @pH 8.5 (made in step 7 above) into the microcentrifuge tube containing the filter paper 'ear'
SECTION: Wash Extracted DNA from filter paper
8.2. ensure the filter paper ear is completely submerged in the liquid, close lid, vortex thoroughly for 20 seconds
SECTION: Wash Extracted DNA from filter paper
8.3. Ensure the filter paper ear is submerged following vortex, with lid closed, place tube on rocking platform at 4C, 500rpm for 2 hours
SECTION: Wash Extracted DNA from filter paper
8.4. following 2 hours of rocking, the DNA will be suspended in the eluate in the tube. 
SECTION: Polymerase Chain Reaction
9. Prepare a clean and sterile bench space for PCR
SECTION: Polymerase Chain Reaction
9.1. Prepare mastermix following the protocol for the  QiagenTaqPCR Master Mix Kit (Catalog #201445). 

MasterMix:

 

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
SECTION: Thermal Cycle
9.2. 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
SECTION: Gel Electrophoresis
10. 2% Agarose Gel Electrophoresis with Biotium Gel Red