Introduction
This document provides general instructions for sampling only fish fin clip tissue in 96-format plates for DNA extraction at Intertek after DNA extraction optimization has been completed.
Intertek requests 2-4 plates of dummy (i.e., non-precious and expendable tissue) collected and prepared in a feasible workflow to establish a preferred method to prepare samples for extraction going forward. Taken into consideration are tissue type, tissue amount, and preservation method.
If other types of tissue or samples are desired, discuss the matter in advance with your BI coordinator. Additional instructions may apply.
Communicating with Intertek
All email communication with Intertek must be addressed to Agritech Sweden (agritech.sweden@intertek.com) with bi-genotyping@cornell.edu in the CC field. You must alert Intertek via email at least 15 days prior to sending samples, in order to ensure the fast turn-around time. For larger sample volume (>10,000 samples), please submit the order at least 25 days prior to sending samples. To make the alert announcement, attach a draft order form in an email message to Agritech Sweden (agritech.sweden@intertek.com) with bi-genotyping@cornell.edu in the CC field.
Required plasticware
Preferred products 96-well plate format: AbGene 1.2 mL 96-well polypropylene storage plates (AB0564) 96-well cap format: AbGene 96-well plate mats (AB0674) Backup products (first choice) 96-well plate format: Costar Corning 1 mL 96-well deep-well polypropylene blocks with round bottom (Corning Life Science product number 3959) 96-well cap format: Costar Corning 1 mL 96-well block sealing mats (Corning product number 3080) Backup products (second choice) 96-well plate format: Corning 1.2 mL 96-well cluster tube system with tubes in strips of 8 (07-200-317) 96-well cap format: Costar 1.2 mL 96-well cluster tube system with caps in strips of 8 (07-200-323)
How to prepare and collect samples
1. Design the genotyping experiment and determine the number of plates needed (number of samples/94), striving for complete plates in sets of 4.
Best pricing is achieved at 16 plates per order.
Consider adding more samples to fill out any partial plates. A full plate is 94 samples.
2. Send an email to bi-genotyping@cornell.edu with your plate count request and a brief summary of your experiment.
3. Select the fish to be sampled. Label (with PIT tags) before you sample. Poor quality samples from old, diseased, or senescent tissues lead to low quality results.
4. Create your sample list ahead of time. A partially-filled Intertek order form is available below.
5. Pre-label all your empty sample plates with human-readable text. Include barcodes if possible.
Always leave wells G12 and H12 empty in all plates. They are used by DArT for lab controls during sequencing.
Documentation is essential to link the sample IDs to the fish PIT tag IDs.
6. Prepare the sampling tools, sample list, and labeled plates. (Be sure your other logistics are in order before you start sampling.)
Various sampling devices can be used as long as the samples are easily identified, and uniform sampling is maintained for all samples.
Scissors or side-cutters are usually all that is necessary to acquire fin clips, though serrated fly-tying scissors are ideal.
Sampling tools should be disinfected between collections with an 80% EtOH bath to eliminate potential disease transfer and DNA contamination.
7. Keep the samples and plates cool on wet ice while sampling.
8. Select the tissue (developmental stage and position) to be sampled .
Fin clips, usually from the caudal fin, are ideal for non-lethal tissue collection for genetic analyses. Alternatively, adipose fins can be used but as the tissue does not regenerate, the fish is left without an adipose fin for the remainder of its life, which might be important for mate selection in wild populations (e.g., brown trout). Pelvic fins are not recommended to their small size on young fish. Caution Avoid cross-contamination at all steps. Clean the sampling tool before you start sampling and thereafter every sample. If a fish is missing, or can't be sampled, then mark the well on the plate and make a note in the sampling sheet as this helps avoiding mis-sampling. Wells for a missing sample can be marked by coloring the edge of the well with a marker pen or by placing a temporary cocktail stick or toothpick that is removed when sampling is completed.
9. Collect samples of the appropriate amount and age, based on your DNA extraction optimization test.
Clip a piece of tissue ~1 cm2 off the top of the caudal fin. This will generate enough DNA to allow for repeated genetic test from the same tissue sample.
Do NOT sample tissue types or amounts that deviate from the agreed upon protocol with Intertek based on DNA optimization testing. Too much tissue or new tissue types will hinder thorough drying/grinding and will decrease the amount and quality of extracted DNA.
For specific projects, other amounts of tissue outside of the recommendation can be agreed upon prior to sampling.
10. Preserve the samples.
Add diluted ethanol to each tube, submerging the sample.
Preferred for best quality extractions: 90-95% ethanol diluted with distilled water
Acceptable for minimal quality extractions: 80% ethanol diluted with distilled water.
11. Seal the plates with Intertek-approved caps or mats.
Be sure to press down on the seal to secure it fully and completely on the plate.
If tubes are used, apply a lid on the box and affix this with the clips and/or rubber bands. All plates can then be wrapped in (a) plastic bag(s) and sealed with tape or rubber bands.
If possible, use parafilm or other liquid barrier to double seal the tubes or sealed plates.
12. Request BI’s approval to ship plates (if BI is paying for genotyping) by sending an email to bi-genotyping@cornell.edu with the following information:
Your completed order form
Any information about sample ploidy or wild origin
A photograph of your plate labels
Caution. Failure to receive prior approval by BI will cause processing delays, increase cost to you, and may flag your order for destruction.
13. BI will apply the appropriate quote number and PO number. BI will double check that all required details are provided and approve the plates for shipping.
14. Print the completed Intertek order form that you receive back from BI.
15. Insert into an outer package a) the printed order form and b) the sealed plates.
The outer package can be a cardboard box.
An inner package, such as a styrofoam, box can be used to prevent plate breakage during shipment, but is not required.
Do not send plates on dry or wet ice. It is not necessary for fully dried leaf tissues and is more likely to moisten samples rendering them unusable.
16. Ship the entire package to the Intertek lab via courier according to the instructions below “How to send samples”.
17. Email the completed Order Form to Intertek and include the courier and tracking information.
18. Alert Intertek that an order is incoming by emailing a draft order form to Agritech Sweden (agritech.sweden@intertek.com) with bi-genotyping@cornell.edu in the CC field.
How to send samples
1. Intertek recommends the courier DHL for the fastest logistics. However, other couriers can be used.
2. Fill out the Commercial Invoice for your chosen international shipping company.
Requirement: Use Commodity Code 051191 for samples of “animal products not elsewhere specified or included; dead animals of chapter 1 or 3, unfit for human consumption: Products of fish or crustaceans, molluscs or other aquatic invertebrates; dead animals of chapter 3”.
3. Create a Pro-forma document on your institution’s letterhead with the following statement: “For analytical purposes only, not for breeding, will be destroyed.”
Pro-forma document template: |
4. Attach the Commercial Invoice, the Pro-forma document, and the shipping manifest to the outside of the parcel.
Caution
You MUST write the correct Commodity Code on the Commercial Invoice.
You MUST include the statement “For analytical purposes only, not for breeding, will be destroyed” on the Pro-forma document.
You MUST attach all paperwork on the outside of the parcel.
5. Send the parcel by courier to the following address:
INTERTEK SWEDEN |
|
Elevenborgsvägen 2 | Phone: +46 40 69 28 001 |
230 53 Alnarp Sweden | Email: agritech.sweden@intertek.com (CC bi-genotyping@cornell.edu) |
6. You will receive an order number and confirmation by email when samples have arrived at the destination.
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Breeding Insight is funded by USDA at Cornell University. For more information, please visit http://www.breedinginsight.org.