Submission Protocols and Pricing

If you have less than 32 samples, standard tube sizes are fine: 1.5 ml, 0.5 ml, 0.2 ml. Please make sure your tubes are completely sealed and labeled on top with your initials and unique numbers. No tape, please! If you have 32 or more samples, please submit them in a skirted (full or semi) PCR plate, and please send a spare plate of the same type with the same seal for balancing the centrifuge the first time you submit samples in a plate. Load your samples by numbers, not letters - in rows of 8 starting with 1. Along with your plate, please send a digital copy of your sample names with associated well positions.

Leave your samples in the freezer in AgC 5023 and submit sample information including number of samples, a digital copy of sample names and what services you require, by filling out the Sample Submission Form.

Templates Requiring Sequencing Reaction

purified PCR product:
Please do not use TE to elute or resuspend DNA! Use purified (PCR) water.

If PCR product is <500 bp: 10-20 ng PCR product + 10 pmol of 1 primer in water: total volume 15 µl.
If PCR product is >500 bp: 20-30 ng PCR product + 10 pmol of 1 primer in water: total volume 15 µl.

plasmid:
300 ng plasmid template + 6 pmol primer in water: total volume 15 µl.

These are guidelines - some, especially longer, templates will require more DNA.

If the annealing temperature of your sequencing primer is not between 50 and 60º C, please let me know in advance! Per request I'm providing a link to my sequencing reaction protocols and more detailed information about DNA quantity.

Sequencing Reaction for Capillary Run

Reactions must be purified (EtOH precipitation or some other dye terminator removal method) and dry (precipitated and dried or lyophilized). If you have 32 or more samples, please submit them in a skirted (full or semi) PCR plate, and please send a spare plate of the same type with the same seal for balancing the centrifuge the first time you submit samples in a plate. Load your samples by numbers, not letters - in rows of 8 starting with 1. Along with your plate, please send a digital copy (Excel or text file) of your sample names with associated well positions.

Submission of Samples for Genotyping

Please obtain a plate and septa seal compatible with the AB 3130xl (plates and seals are provided by NAEF if you are using our genotyping services) and load 1 µl/well (usually) of your samples in groups of 16 starting with rows 1&2. Add 9 µl of Hi-Di Formamide containing the appropriate amount of labeled size markers to each well containing sample and add 10 µl Hi-Di to wells in each group of 16 if they don't contain sample. I’ll denature them just before I put them on the sequencer. If you denature them yourself as well, please disable any heated lid on your thermal cycler and leave it up off of the plate. The septa seals are designed to allow capillary penetration and are not recommended for thermal cycling.

Please provide information for the run(s) including the dyes/dye set and size standards used. If you would like your sample names (one per well) to appear as the file names for your genotyping files, please provide a digital version of the well positions and sample names. If no sample names are provided I will name the files with their well positions.

I can work with you on permutations of these procedures. If you have any questions at all, please don’t hesitate to contact me!

All submissions must be labeled with your initials and unique numbers! For example: If George Washington Carver submitted samples, they would be labeled GWC1, GWC2, etc.

Pricing

Sequencing reaction/ cleanup/ capillary run: $10.00
User provides purified DNA (PCR product or plasmid) and primer; NAEF will do the sequencing reaction, dye-terminator removal and capillary run

Run ready (sequences or fragments): $1.00
User performs the PCR or sequencing reaction and dye-terminator removal or loading of genotyping samples and Hi-Di Formamide into the appropriate plate; NAEF will do the capillary run

Project bulk discounts may be available. Please e-mail the Director at fungi@uwyo.edu for more information.

Use of NAEF equipment and facilities:

Use of the Applied Biosystems 7500 Real-Time PCR System will be charged based on usage time per PCR reaction (plate or set of tubes). Nonspecific reagents and kits will be made available at my cost – please let me know what you need. All users must be “checked out” on software and procedures. Optical plates and seals are available from NAEF.

The NAEF Teaching/Prep lab is a fully equipped lab available for your use free of charge provided you submit your samples for sequencing and genotyping to the NAEF Service Core lab! Refrigerator and/or freezer space is available.
Access to the lab is through a card lock using your campus ID. All users of the facility will be “checked out” on the use of the equipment and laboratory procedures and etiquette. For access, please contact Terry McClean. Access will be recorded for all users and activated/deactivated at my will.