IRRI SERVICE LABORATORIES
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3.A Submit samples to GSL
3.A Submit samples to GSL
• Submit DNA samples
• Submit DNA samples
GSL only accepts DNA for the 7K Infinium genotyping platform. DNA extraction is included in the other genotyping platforms (i.e. trait genotyping, 1k-RiCA, QC genotyping).
GSL only accepts DNA for the 7K Infinium genotyping platform. DNA extraction is included in the other genotyping platforms (i.e. trait genotyping, 1k-RiCA, QC genotyping).
3.1 Prepare sample quality & quantity
3.1 Prepare sample quality & quantity
Prepare 100ng/µL and 50 µL for the DNA sample concentration and volume respectively. All samples must be RNAse-treated and have a nanodrop reading of 1.8-2.0 at 260/280 to pass the quality check.
Prepare 100ng/µL and 50 µL for the DNA sample concentration and volume respectively. All samples must be RNAse-treated and have a nanodrop reading of 1.8-2.0 at 260/280 to pass the quality check.
Note: DNA extracted through quick & dirty methods (e.g.TPS) are not accepted due to its effect on data quality.
3.2 Plate the DNA samples
3.2 Plate the DNA samples
Your samples must be submitted in a 96-well polypropylene plate, arranged column-wise to match the layout in your GSL Request form.
Your samples must be submitted in a 96-well polypropylene plate, arranged column-wise to match the layout in your GSL Request form.
3.3 Label the plates
3.3 Label the plates
Each plate must be labelled according to the format below.
Each plate must be labelled according to the format below.
GSL Code Name_Name of Requestor_Plate #_Date of Sample Preparation (DD-MMM-YYYY)
GSL Code Name_Name of Requestor_Plate #_Date of Sample Preparation (DD-MMM-YYYY)
Example: 2020-1_INF_JLopez_Plate 1_05May2020
Example: 2020-1_INF_JLopez_Plate 1_05May2020
3.4 Dry the samples
3.4 Dry the samples
Submission of DNA in aqueous or solution form is discouraged, (especially if dry ice is not provided) while freeze-dried or dried samples are preferred. You may dry your samples using a plate-based speed vac or perform the PCR instructions below.
Submission of DNA in aqueous or solution form is discouraged, (especially if dry ice is not provided) while freeze-dried or dried samples are preferred. You may dry your samples using a plate-based speed vac or perform the PCR instructions below.
1. Place your samples in a half-skirted plate and seal them properly.
1. Place your samples in a half-skirted plate and seal them properly.
2. Spin your samples in a centrifuge to ensure that all liquids are at the bottom of the wells with no air bubbles.
2. Spin your samples in a centrifuge to ensure that all liquids are at the bottom of the wells with no air bubbles.
3. Place your samples in a thermocycler, remove the seal, and heat it at 65°C with the lid open until the DNA is completely dried.
3. Place your samples in a thermocycler, remove the seal, and heat it at 65°C with the lid open until the DNA is completely dried.
Note: Drying 30 µL will take 1.5-2.5 hours
Warning: Do not heat your samples over 65°C due to the risk of degrading the DNA.
Tip: You may determine the time needed to dry the DNA by performing a trial run using water samples (or 1xTris-EDTA) with the same volume as your DNA samples.
3.5 Prepare samples for shipping
3.5 Prepare samples for shipping
Prior to shipping samples, ensure that a signed Letter of Engagement (LOE), an accomplished GSL Request Form, and a list of materials have been emailed to and approved by GSL.
Prior to shipping samples, ensure that a signed Letter of Engagement (LOE), an accomplished GSL Request Form, and a list of materials have been emailed to and approved by GSL.
1. Ship your samples as either dried/freeze-dried pellets or dried precipitates.
1. Ship your samples as either dried/freeze-dried pellets or dried precipitates.
2. Include backup DNA in case a replacement is needed. Since these are sample replicates, you don’t need to indicate them in the GSL Request Form.
2. Include backup DNA in case a replacement is needed. Since these are sample replicates, you don’t need to indicate them in the GSL Request Form.
3. Secure the plate of samples by placing it between two pieces of cardboard.
3. Secure the plate of samples by placing it between two pieces of cardboard.
Warning: Shipping DNA in aqueous or solution form is highly discouraged especially if dry ice is not provided.
4. Encase the plate and cardboard pieces in bubble wrap to prevent any physical damage.
4. Encase the plate and cardboard pieces in bubble wrap to prevent any physical damage.
5. Transfer the encased plates in a zip lock bag to contain any spills.
5. Transfer the encased plates in a zip lock bag to contain any spills.
• Submit leaf samples
• Submit leaf samples
5.1 Collect the leaves
5.1 Collect the leaves
1. Collect two leaves per sample with length and width dimensions of 152.4 mm (6.0 in) by 6 mm (0.24 in) respectively.
1. Collect two leaves per sample with length and width dimensions of 152.4 mm (6.0 in) by 6 mm (0.24 in) respectively.
Warning: You should only collect leaf samples from non-diseased plants 20-35 days after transplanting.
2. Place the leaves in ¼-size glassine bags with lengths and width dimensions of 51 mm (2.0 in) by 145 mm (5.71 in) respectively.
2. Place the leaves in ¼-size glassine bags with lengths and width dimensions of 51 mm (2.0 in) by 145 mm (5.71 in) respectively.
3. Label each glassine bag with the same Sample IDs provided in the GSL Request Form.
3. Label each glassine bag with the same Sample IDs provided in the GSL Request Form.
4. Immediately after collecting the leaves, place them in a ziplock bag and ice cooler to prevent the leaves from rolling due to the heat.
4. Immediately after collecting the leaves, place them in a ziplock bag and ice cooler to prevent the leaves from rolling due to the heat.
5.2 Prepare samples for shipping
5.2 Prepare samples for shipping
1. Dry your samples either by using a freeze-dryer (preferred) or by placing them in a 50°C oven overnight.
1. Dry your samples either by using a freeze-dryer (preferred) or by placing them in a 50°C oven overnight.
2. Bundle or group together the glassine bags according to the sequence indicated in the GSL Request Form. Treat each bundle as one plate.
2. Bundle or group together the glassine bags according to the sequence indicated in the GSL Request Form. Treat each bundle as one plate.
3. To avoid moisture build-up, place the bundle with silica beads or any desiccants in a zip-lock plastic bag.
3. To avoid moisture build-up, place the bundle with silica beads or any desiccants in a zip-lock plastic bag.
4. Place all the samples in a container/box and include the following documents:
4. Place all the samples in a container/box and include the following documents:
- Phytosanitary Certificate from the country of origin
- Import Permit (IP) / Plant Quarantine Clearance.
- Signed Letter of Engagement
- Accomplished GSL Request Form
Note: GSL requires 2-4 weeks to process and email you the IP, which defines the allowable weight of samples you can import to IRRI. The weight stated in the IP must coincide with the weight in the Phytosanitary Certificate.
• Submit seed samples
• Submit seed samples
5.1 Identify the appropriate seeds for sampling
5.1 Identify the appropriate seeds for sampling
Samples should be taken from seeds that are filled, non-diseased, and were harvested no longer than 2.5 months beforehand.
Samples should be taken from seeds that are filled, non-diseased, and were harvested no longer than 2.5 months beforehand.
5.2 Collect the seeds
5.2 Collect the seeds
1. Collect two grams of seeds per sample (threshed). Alternatively, a single panicle of seeds can be collected.
1. Collect two grams of seeds per sample (threshed). Alternatively, a single panicle of seeds can be collected.
2. Place the seeds in #4 coin envelopes with lengths and width dimensions of 231 mm (9.1 in) by 145 mm (5.7 in) respectively.
2. Place the seeds in #4 coin envelopes with lengths and width dimensions of 231 mm (9.1 in) by 145 mm (5.7 in) respectively.
3. Label the bags using the GSL-provided barcodes or any label which indicate the same sample names in the GSL request form.
3. Label the bags using the GSL-provided barcodes or any label which indicate the same sample names in the GSL request form.
4. Bundle or group together the glassine bags per plate according to the sequence indicated in the GSL request form.
4. Bundle or group together the glassine bags per plate according to the sequence indicated in the GSL request form.
After GSL reviews your samples, you will be sent a confirmation email that informs you whether your samples have been accepted or rejected. If accepted, then you may proceed to ship your samples. If rejected, then GSL will provide guidelines on how to resubmit samples.
After GSL reviews your samples, you will be sent a confirmation email that informs you whether your samples have been accepted or rejected. If accepted, then you may proceed to ship your samples. If rejected, then GSL will provide guidelines on how to resubmit samples.
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