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Sample Submission Requirements

Sample Submission requirements for AGRF services:

Microsatellite Genotyping

The amount of DNA required for submitting samples to AGRF for Microsatellite Genotyping is dependent on the number of markers to be screened.

The quantity of DNA required corresponds to at least twice the amount required, as any sample or marker which fails in the original round will be repeated once.

15ng of DNA will be used for each PCR reaction. For example, a screen comprising 14 markers: 14 x 15ng x 2 = 420ng or 42ul at 10ng/ul of each sample is required.

If submitting more than 24 samples the AGRF prefer DNA samples to be sent in clearly labelled 96-well plates. Please submit your samples in a securely sealed plate according to the plate template.

Please leave positions A1 and H12 blank for internal controls. Also leave A2 blank for mouse sample submisions.

If your DNA source is other than human, please provide additional positive DNA controls.

Fragment Analysis

For Fragment separation, a 10ul aliquot or pellet of PCR product is required.

If submitting more than 24 samples, the AGRF prefer samples to be submitted in clearly labelled, sealed 96-well plates according to the plate template.

Genome-Wide SNP

Affymetrix and illumina

In order to achieve optimal results, these systems require high quality DNA. Samples should be provided at 100ng/ul in reduced EDTA TE Buffer (10mM Tris HCL, 0.1mM EDTA, pH 8.0).

For the Affymetrix 10K, 50K or 250K service, the minimum amount of DNA required is 1ug i.e.10ul at 100ng.

For the Affymetrix 100K, 500K 5.0 or 6.0 service the minimum amount of DNA required is 2ug i.e.20ul at 100ng.

DNA should be high quality: 260/280 = 1.7 - 1.9, 260/230 = 1.6 - 2.2. Samples should also show no degradation of HMW DNA on a 2% agarose gel.

Whole Genome Amplification Samples

For Affymetrix: Whole genome amplified (WGA) samples should be provided undiluted; the minimum volume required is 50ul. The AGRF recommends samples should be amplified with the Qiagen REPLI-g kit; no product cleanup is required.

For illumina: Illumina does not recommend using WGA samples as input for the Infinium Assay as the first step in the protocol is a WGA step. Up to a 4% decrease in call rate was observed in the limited internal testing performed. The decrease in call rate will vary depending on the specific sample and WGA method used. Additionally, any allelic bias present in the original WGA sample may be compounded by another WGA reaction.

All samples will be assessed internally by the AGRF using the NanoDrop and an agarose gel. If any DNA samples is deemed sub-optimal at the time of submission, AGRF will advise the client and then provide the opportunity to re-submit a new sample.

Targeted / Functional SNP Genotyping

Affymetrix

In order to achieve optimal results, the Affymetrix system requires high quality DNA. Samples should be provided at 100ng/ul in reduced EDTA TE Buffer (10mM Tris HCL, 0.1mM EDTA, pH 8.0).

The minimum amount of DNA required is 1ug i.e.10ul at 100ng.

DNA should be high quality: 260/280 = 1.7 - 1.9, 260/230 = 1.6 - 2.2. Samples should also show no degradation of HMW DNA on a 2% agarose gel.

Whole genome amplified (WGA) samples should be provided undiluted; the minimum volume required is 50ul. The AGRF recommends samples should be amplified with the Qiagen REPLI-g kit; the appropriate product cleanup will be performed by the AGRF.

Custom SNP

Sequenom Platform

SUBMITTING DNA SAMPLES:

All samples should be normalised to the same concentration of approximately 10 ng/ul.

For most projects, 10-20ul at a concentration of at least 10ng/ul of DNA is acceptable. Higher concentrations may be sent provided all samples are standardised to approximately the same concentration. Due to the DNA required for Quality Control and assay design, larger projects ( >50 SNPs) will require a larger amount of DNA. Please contact us to confirm quantity required.

If DNA is quantified by OD, acceptable A260/A280 ratios are between 1.6 and 2.0.

Please elute with either purified water or diluted Tris HCl. High concentrations of EDTA may adversely affected our processes, so please avoid this if possible.

SAMPLE FORMAT:

Samples should be sent in 96-well format.

Our robotics and downstream processes are configured to handle V bottom plates, where most commercially available varieties are acceptable. Please refrain from submitting in flat or round bottom wells. If these are necessary, a greater volume of DNA will be required.
Half skirt of full skirt plates should be used as they tend to be more robust as well as providing space for plate labeling. Chimney stack plates should be avoided since they cannot be affectively sealed.
Make sure you use a correct seal with a plate to avoid evaporation.

Please submit your samples in vertical format. This means applying your samples from A01 down to H01, then from A02 down to H02 and so on. Wells left as blanks in between samples will be billed. This does not include blank wells that follow the final sample on the plate. If you have already placed your samples in horizontal order and can not change it, please contact us.

PLATE SEALS

Adequate seals must be placed on all submitted plates to avoid cross-contamination. For most plates, a PCR film or tape is preferred compared to strip caps, although you may still use these if packaged with extra care. Alfoil seals are not acceptable.

Upon delivery, if DNA isolation has been compromised by incorrectly sealed plates, the AGRF will not begin the project until further discussion with the client.

If you are sending plates of suspended DNA, an incorrectly sealed plate may also be susceptible to evaporation. This will alter the concentration and amount of DNA added to each reaction and may possibly affect the results.

Heat Sealing machines can also be used, but please make sure the sealing tape is an easy peel type that can be removed without difficulty.

A recommended seal is the ABgene Adhesive PCR Film: AB-0558, though most tape pads are sufficient if securely applied.

Please enclose plates in a resealable plastic bag.

SAMPLE LABELLING:

Please label all your plates clearly with:

Plate name/ number

Company name

Submitters’/ project leader name

Date

This will help us better to identify your plates more readily should you require them at projects’ completion.

TRANSPORT of DNA to AGRF

Transport is entirely dependent on the client’s discretion. HOWEVER the method of transport can be detrimental to the quality and safety of your DNA. Depending on whether DNA is sent to us in either dry pellet or suspended form will determine the optimal method of delivery.

1. Dried Down DNA:

AGRF recommends that samples are sent dried down as this is the easiest and safest way to transport DNA. By drying the samples down, evaporation of the sample is eliminated. It also reduces possible cross contamination if your plate experiences rough transport as well as avoiding the need for sending plates on dry/standard ice.
Please note: If drying the sample down it is necessary to send 200ng so it can be resuspended in 20ul of water to make 10ng/ul.
Plates should be wrapped in bubble wrap or similar and sent via express post overnight.
Please be advised that although a semi skirt plate for DNA is recommended by AGRF, this allows the exposed bottom of the wells to be vulnerable to possible breakage if the plate is not wrapped correctly. If more than one plate is being sent, the plates should be positioned with the wells facing each other to protect them from damage, then securely encase them in Bubble Wrap.

2. Suspended DNA:

For clients within a distance of Melbourne to Brisbane, sending samples with frozen ‘cold packs’ is sufficient. Plates should always be wrapped in bubble wrap, or something similar, and sent via a transport company of your choice, eg. a courier, or express post.
For customers that are a further distance away it is recommended to send DNA with ice or dry ice in an esky. Remember to protect your plates by filling the box with stuffing so your plates are safely constricted and are not able to move too much.
For customers that are overseas, AGRF strongly recommends that samples be sent frozen with dry ice. This will protect the integrity of the DNA and avoid evaporation. Be reminded that a clearly marked air hole must be placed for the dry ice to breathe so as not to explode the container.

For any inquiries or problems please contact us.