1.Create Order

• Register, if not already, then Login to Online Ordering at  https://ordering.seqart.net/cgi-bin/order/login.pl
• Create a Sample Tracking Template File (required next);

 Remember wells G12 and H12 are used for control and MUST BE EMPTY

• Create an order – Select ‘Order Services’ to access Online Ordering and create your order;
• Print a copy of the Service Specification to include with order package.

   2.GENOMIC DNA (GDNA) QUALITY AND QUANTITY REQUIREMENTS

For DArT genotyping assays we recommend 40-50 ul of restriction enzyme grade genomic DNA (gDNA), resuspended in aqueous solution as EB buffer at a concentration of 50–100ng/µl. Other acceptable solutions are Molecular Grade water and TE buffer with lowered EDTA concentration.

If you have less gDNA than recommended quantities, please contact us at support@seqart.net. We may still be able to process your samples as we successfully produced genotyping data from samples with concentration less than 10ng/µl.

DArT assays tolerate well up to five-fold differences in gDNA concentration, however the more uniform gDNA concentration of submitted samples the better quality of genotyping data.

Please be aware of additional fee incurred if gDNA concentration variation exceeds acceptable levels (up to 5-fold) and we need to adjust the gDNA concentration before the carrying out the assays.

We use 2µl gDNA of recommended quality/quantity per assay, therefore:

•  for organisms/products listed in our system please submit at least 30µl of gDNA of required concentration, although 40µl is recommended.
•  for new organisms/products please submit minimum of 40µl of gDNA of required concentration, however higher volumes up to 50µl are welcome.

In any case do not exceed 70µl gDNA as it significantly increases the risk of sample cross-contamination during shipment and handling.

As for all genotyping methods, gDNA quality has a major effect on genotyping data quality. Therefore, we recommend performing gDNA ‘mock incubation’ test of the samples before shipping them to us, even if it is on a subset of the samples. Please perform ‘mock incubation’ test as follows: incubate 2µl of gDNA in Restriction Enzyme buffer at 37°C for 2 hours and then resolve the samples on a 0.8% TAE agarose gel. Good quality gDNA gives a high molecular weight band on the gel.

3. gDNA derived from museum collections/valuable/prolonged storage materials

If you are submitting gDNA derived from museum collections/valuable/prolonged storage materials, irrespective of expected quality/quantity, please ensure you alert us by adding adequate note while placing your order and on the Sample Tracking File in the Comments.

If you require any additional information about your gDNA quality/quantity please contact us at support@seqart.net

Suggested, not automated methods of gDNA extraction can be found below:

This method proved to work well for many plant species: 

https://ordering.seqart.net/files/DArT_DNA_isolation.pdf

There is also alternative method developed by one of our collaborators, which should work equally well: https://www.diversityarrays.com/orderinstructions/extracting-dna-from-plants-alternate/.

Please note that we cannot guarantee that these methods will work for any species.

4. SAMPLE PACKAGING

Please package your DNA in:

• Well-sealed 96-well plate(s),
• FULLY SKIRTED Please for rigidity and convenience of bar-coding. To avoid leaks and cross contamination of samples during shipment we strongly recommend:

1.  Fully skirted, V-shape bottom 96 well PCR plates Twin-Tec from Eppendorf (Product code 0030128648) or
2. Strips of 8 clear FLAT caps from Sarstedt (Product code 65.1998.400.¹)

•  Seal firmly/securely
• Package in a rigid box with ample packing to allow for rough handling in shipment;
• Include the printed copy of the Service Specification. For all courier companies: make sure all our contact details are correctly and legibly written, including our phone number.

¹Sarstedt discontinued strip caps 65.989.002 (made in Germany) and 65.1998.002 (made in the USA). These are replaced by 65.1998.400 (made in Great Britain or Hungary) these can be used for the strip chains and the Eppendorf twin Tec 96 well PCR plates.

5. Ship to Address

JACKLINE CHEPKOECH

SEQART AFRICA,
International Livestock Research Institute (ILRI) Campus, Old Naivasha road,

P.O Box 30709, Nairobi 00100, Kenya

Tel: +254 20 422 3898