USDA Genotyping Lab Tissue Collection

USDA Genotyping Lab – Tissue Collection (Updated July 2012, Fargo, ND)

Breeders should contact the genotyping lab (see below for contact information) before samples are ready to be collected for genotyping. The 96-deep-well blocks and the block labels will be supplied to the breeders. Each block will have a fixed amount of silica gel desiccant. The consistent amount of the silica gel added in the blocks will facilitate the block processing using the robot in the genotyping lab. The block label includes the information of breeder code, year, and block number. The label will facilitate the tracking and data points tallying of the genotyping jobs processed in the lab.

Leaf sample collection in 96-deep-well blocks filled with silica gel. The use of silica gel eliminates the need for liquid nitrogen or freeze drying. (Revised Feb.2011)

1. If the blocks are received before tissue is ready to be collected, they can be stored in an oven at 65oC. Be sure the blocks are not touching one another as they can melt together and will cause problems on the tissue grinder.

2. To collect samples, (i) orient the block with the A01 well to the upper left and H12 well to the lower right, (ii) remove the cap-mat from one block at a time, and (iii) collect 1.5′′ to 2′′ leaf segment preferably at seedling (3-leaf) stage, fold it and insert it in the well. The amount of silica gel added was calibrated and is enough to dry one piece of leaf segment within a few days. Note: If too many leaf segments are inserted, they will either get moldy or take a very long time to dry, thus affecting DNA quality. Tissues should be collected at room temperature. Do NOT place the silica filled block on ice while collecting as this will affect tissue drying and DNA quality.

3. Insert the samples starting at the top of a column and moving down (see figure). Well positions A01 and B01 are reserved for parents/checks. If more checks are used to generate the population, they can go into wells C01, D01, etc. Well H12 should always be left empty to help with orienting the block and for adding the genotyping control. Well H12 will have blue indicator silica in it; if the indicator silica turns pink, the silica has been hydrated and the tissue will not dry properly.

4. Put the cap-mat back on the block after finishing all wells, and press it tightly to seal the wells. Invert the block a couple of times to ensure that the leaf tissue is in contact with the silica gel. Leave the block on the bench at room temp to dry. Blocks can be shipped while samples are drying. Samples should be dried within 2 to 3 days, which usually coincides with the time when the indicating silica gel turns pink. Store the blocks at room temp until DNA extraction. Note: The shelf life at room temp is estimated at about 1 month for barley and wheat leaf samples. For long-term storage, it is the best to store blocks at –20oC after samples have been dried.

USDA-ARS Genotyping Protocols 1 2 3 4 5 6 7 8 9 10 11 12 A P1 S7 B P2 S8 C S1 S9 D S2 S10 E S3 F S4 G S5 H S6 Blank Tissue Collection Figure. Please collect tissue starting from the top of a column and moving down; return to the top of the next column and move down again. Include parental tissue in wells A1 and B1, and then continue on with samples. Leave well H12 blank if possible*. *If collecting for Illumina assays, do NOT leave H12 empty.

Silica gel filling protocol (Ref 2)

To fill silica gel to the wells of 96-deep-well plates, use a 96-well round bottom microtitre plate and fill the wells with a silica gel mixture by scooping. An empty deep 96-deep-well plate is then placed upside down on the microtitre plate. The plates are held together and inverted, thus transferring the silica gel from the microtitre plate to the deep-well plate. The amount of silica gel filled in each well is estimated at 0.25 g. Silica gel used is the plain type 6-12 mesh; as a moisture control, the plain type silica gel is mixed with a small amount of the indicating type**. Deep-well plates are sealed tightly with a plate mat to prevent contamination and moisture entering wells. Before collecting samples, if the silica gel indicators turn pink, place the deep- well plates in a 65oC oven to dry until the indicators turn blue. Always leave the cap-mat on the plate to prevent moisture getting in the wells and causing silica gel to lose its desiccating function.

**Care should be taken when using the blue indicating type, using too much can cause DNA to degrade during DNA extraction in the presence of silica gel.

Company Order # Product Unit Price SEAHORSE - 96 Well 2ml Phenix MPI-009 Deep Well Plate, PP, Pyramid 25pk 82.90 Bottom, Square Square Silicone Sealing Mat Phenix MPI-P07 Case-100 290.90 for deep well Silica gel, 6-12mesh, non- VWR 26668-112 Each 54.97 indicating (Eagle Chemical) Silica gel, 6-12mesh, VWR 26668-121 Each 37.87 indicating (Eagle Chemical)

USDA-ARS Genotyping Protocols Reference. 1. Chase, M.W. and D.M. Hillis. 1991. Silica Gel: An ideal material for field preservation leaf samples for DNA studies. Taxon 40:215-220. 2. Bodo Slotta, T.A., L. Brady and S. Chao. 2008. High throughput tissue preparation for large-scale genotyping experiments. Mol. Ecol. Resources, 8:83-87.

Contact Information Shiaoman Chao 701-239-1462 [email protected] Mary Osenga 701-239-1467 [email protected] Fax 701-239-1445

Mailing Address USDA-ARS-BRL Genotyping Lab 1605 Albrecht Blvd N Fargo, ND 58102-2765

USDA-ARS Genotyping Protocols