TITLE: Preparation of Tissue Samples for DNA Extraction and Copy Number Analysis by iDNA Technologies
AUTHORS: Lynn Doran

[DESCRIPTION]
Preparation of leaf tissue for shipment to iDNA Genetics for DNA extraction and copy number analysis.  

NOTE:  TOBACCO TISSUE IS REGULATED AS DRUGS AND ALCOHOL.  iDNA maintains special permits that allow the import of small quantities of tobacco tissue for destructive analysis only.    Before sampling tobacco tissue, contact iDNA for up to date required forms and special shipping instructions not contained in this protocol.   As of 6/11/2024, iDNA Technologies contacts are michala.woodvine@idnagenetics.com and gelli.christodoulou@idnagenetics.com.

Alternatively for tobacco samples, extract DNA using Qiagen DNeasy Plant Kit and ship per protocols.io protocol "Preparation of DNA Samples for Copy Number Analysis by iDNA Technologies".

For all soybean, cowpea, miscanthus, sorghum, maize, either tissue or DNA can be sent if iDNA has established primers for analyzing copy number.  However, iDNA prefers to receive tissue when possible.

[STEPS]
SECTION: Get a Quote
1. Email iDNA Technologies for a quote.  Include the number of samples to be tested, which genes you want copy number analysis on, and which species the samples are from. 

Include one sample with a known copy number of 2 as an internal reference standard for each assay (gene).
 

 

SECTION: Get a Quote
2. When you've received the quote, enter it into the appropriate lab group's Quartzy (or other purchasing request platform) account.

Enter the following information:
Vendor: i-Dna Biotechnology
Catalog #: xxxxxxxxx
Unit size: 1
Qty: 1
Unit Price: From quote (convert from British pound to US dollar before entering)
Upload File: Attach quote from iDNA
Grant ID: Appropriate CFOP for project
SECTION: Sample Tissue
4. Sample three to four #1 Humboldt cork borer leaf punches (4 mm) from the most recently developed leaf by pressing the cork borer through the leaf onto a rubber stopper and using a twisting motion.

 
 
SECTION: Sample Tissue
5. Push the leaf discs out into the bottom of its pre-labeled collection microtube.
 
SECTION: Sample Tissue
6. Repeat for all desired samples.  
SECTION: Sample Tissue
7. Store collection tubes on ice while sampling.  Tubes can be at room temperature for short periods of time (i.e. while you are sampling a strip or box of tubes).  

 

 
SECTION: Sample Tissue
9. Stretch a sheet of parafilm across all tubes in a rack.
SECTION: Sample Tissue
10. Poke pin holes using a needle or 10 ul pipette tip in the parafilm over each tube.
 
SECTION: Sample Tissue
11. Cover the tubes in the rack with their lid.
SECTION: Sample Tissue
12. Freeze at -80C overnight.

 

SECTION: Freeze Dry Samples
18. Remove samples from freeze dryer desiccator attachment, remove parafilm, and cap tubes using strip caps.

 

 


SECTION: Freeze Dry Samples
13. Prepare freeze-dryer by pre-cooling and vacuuming to equilibration based on manufacturer’s and lab manager's instructions.
SECTION: Freeze Dry Samples
14. Transfer tubes from -80C to freeze dryer and lyophilize at <-40C and <0.5 mbar overnight.  If a large number of samples will be prepared or if it is easier to keep samples in the racks during freeze-drying, a vacuum desiccator can be added to the freeze-dryer to increase capacity up to 3 full collection tube racks.

SECTION: Sample Tissue
3. Pre-label the collection tubes.

 

 
SECTION: Freeze Dry Samples
19. Replace tube rack lid and tape closed.
 

SECTION: Prepare Samples for Shipment
20. Clearly label the side of the box, on the box or tape, with the lab name, date, sample tissue type, and gene requested for copy number.
SECTION: Prepare Samples for Shipment
22. Weigh the sample box and record the weigh on the commercial invoice form.  The number of packages refers to the number of exterior boxes (typically 1).  The quantity refers to the number of items in the package, typically we use the number of sample tubes for this value.  For unit value, we typically assign each sample tube the value of $1.
SECTION: Prepare Samples for Shipment
21. Prepare the shipping documents.
SECTION: Prepare Samples for Shipment
23. Give the package and shipping documents to IGB Shipping Department to review.  If their advice differs from the above, heed their advice instead.

SECTION: Prepare Samples for Shipment
24. Forward shipping tracking information from IGB Shipping Department on to iDNA Genetics.  If you had not previously sent them the completed sample template, "96 well template_iDNA.xlsx", send it with the tracking information.
SECTION: Prepare Samples for Shipment
25. Typically it takes 5-9 days to make it through customs.  If tracking indicates a clearance delay for more than a few days, contact the UIUC/IGB purchaser and/or iDNA genetics to help move the package through export/import clearance.
SECTION: Sample Tissue
8. If sending an internal reference standard, a previously collected sample with a known copy number of 2, add it to the collection tube box from the -80C freezer.

 

SECTION: Freeze Dry Samples
13.1. Review Lab Manager's Quick Start Guide near the instrument.

 

 

SECTION: Freeze Dry Samples
13.4. Press anywhere on the touch screen to wake up the instrument.

 

SECTION: Freeze Dry Samples
13.3. Ensure that all vacuum ports on the manifold are closed.  Vacuum ports are closed when the middle of the flat portion of the port handle is pointing in the opposite direction from the attachment tube.
 

SECTION: Freeze Dry Samples
13.5. Use the touch screen to select "Collector" at the bottom left of the screen.

 

SECTION: Freeze Dry Samples
13.2. Ensure that vacuum manifold is squarely lined up with gasket on top of instrument.

 

SECTION: Freeze Dry Samples
13.6. When prompted, use the touchscreen to press "Start" to start the collector. 
 

SECTION: Freeze Dry Samples
13.7. Wait for the collector to reach temperatures colder than -40C.  The temperature reading on the touchscreen will turn green when the collector has cooled sufficiently.
 

SECTION: Freeze Dry Samples
13.8. Use the touchscreen to select "Vacuum" from bottom left corner.

 

SECTION: Freeze Dry Samples
13.9. When prompted, use the touchscreen to set the pressure to 0.200 mbar and then press "Start" to start the vacuum.  If a hissing noise is audible, recheck that all vacuum manifold ports are in the closed position.  The pump motor should be audible at all times once the vacuum has been started.
 
 
 

SECTION: Freeze Dry Samples
13.10. Wait for the vacuum pressure to turn from "High" to a red number and then from a red number to a green number (~ <0.5 mbar).  When both the collector and vacuum reading are displaying green numbers, the freeze-dryer is ready for use. 

 

SECTION: Freeze Dry Samples
14.2. With the vacuum manifold port still in the off/vent position, push the glass adapter for the desiccator attachment hose into the rubber vacuum tube port.

 

 

SECTION: Freeze Dry Samples
14.3. Ensure the other end of the tube is firmly pushed on to the barb fitting on both sides and on to the attachment port for the desiccator lid.
 

SECTION: Freeze Dry Samples
14.4. For additional room in the desiccator chamber, remove the white mesh shelf and set aside.  Ensure the black gasket that goes between the desiccator and it's lid is present and in good condition.

 

SECTION: Freeze Dry Samples
14.5. Move the frozen samples from the -80C freezer to the desiccator on dry ice.  Remove clear plastic collection box lid and set aside so only vented parafilm remains.  Stack collection boxes in desiccator up to three high.  Replace desiccator lid.

 

SECTION: Freeze Dry Samples
14.6. Open the appropriate vacuum manifold port on the manifold to apply vacuum to the desiccator.  Vacuum ports are open when the middle of the flat portion of the port handle is pointing directly at the middle of the attachment tube.  The vacuum reading on the home screen will go from low pressure/green to "High"/red as the vacuum initially has to remove the air from the desiccator but will return to low pressure/green within 5-10 minutes as vacuum is successfully applied.

 

 

 

SECTION: Freeze Dry Samples
15. Lyophilize at <-40C and <0.5 mbar overnight. 
 

SECTION: Freeze Dry Samples
16. To release the vacuum from the desiccator, rotate the vacuum manifold port handle so that the middle of the flat portion of the port handle is pointing directly at the vent hole, directly opposite the middle of the attachment tube.  A hissing of air being released should be audible.

This will only release the vacuum on the desiccator but will maintain vacuum on the remainder of the freeze dryer manifold.  The vacuum readings should remain green but the lid will lift off the desiccator after the vacuum has been vented, the hissing of air should cease.

 

SECTION: Freeze Dry Samples
17. Shutdown freeze-dryer by warming, releasing vacuum, and removing residual water based on manufacturer’s and lab manager's instructions.

 

SECTION: Freeze Dry Samples
17.1. Press anywhere on the touch screen to wake up instrument.

 

SECTION: Freeze Dry Samples
14.1. Remove the vacuum desiccator from storage in the cabinet below the freeze-dryer.
 

SECTION: Freeze Dry Samples
17.2. Use the touchscreen to select "Vacuum" from bottom left corner.
 

SECTION: Freeze Dry Samples
17.3. When prompted, use the touchscreen to press press "Stop" to stop the vacuum.   The pump motor should no longer be audible.

 

SECTION: Freeze Dry Samples
17.4. Use the touch screen to select "Collector" at the bottom left of the screen.
 

SECTION: Freeze Dry Samples
17.5. When prompted, use the touchscreen to press "Stop" to stop the collector. 
 

SECTION: Freeze Dry Samples
17.6. To start defrost, use the touch screen to select "Collector" at the bottom left of the screen.
 

SECTION: Freeze Dry Samples
17.7. When prompted, use the touchscreen to press "Defrost Options".
 

SECTION: Freeze Dry Samples
17.8. When prompted, use the touchscreen to press "Start" to begin defrosting the collector.
 

SECTION: Freeze Dry Samples
17.9. The defrost will turn off automatically if the chamber reaches 60C.  The amount of water removed from copy number samples prepared as described above is minimal.  The defrost usually turns off automatically in less than five minutes.   If it does not, follow the lab manager's Quick Shutdown Guide.
SECTION: Freeze Dry Samples
17.10. Remove the vacuum manifold and set it on the counter.
 

SECTION: Freeze Dry Samples
17.11. Dry the inside of the collector.   The amount of water removed from copy number samples prepared as described above is minimal and the collector can easily be dried using paper towels.  If a large amount of water is present, follow the lab manager's Quick Shutdown Guide.