Lab Use and Submission Protocols

Non-DNA Molecular Analysis - Core Service and Self Service

Please contact David Perry when starting your project and before preparing your samples.

DNA Sequencing and Genotyping - Core Service

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 version (Excel or text file) with your sample names and associated well positions. 

Leave your samples in the freezer in the Berry Center 320 and submit sample information including number of samples, a digital version of sample names and what services you require, by filling out the online Sample Submission Form.  Please send me your W number from your WyoOne card for access to the Training/Prep Lab, Berry Center 320.

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:
600 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 performed with the BigDye Terminator v3.1 Cycle Sequencing Kit.  (You may use other reaction chemistry from AB, but please check with me first to be sure my instrument is calibrated for your chemistry.)  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 number, not letter - in rows of 8 starting with 1.  Along with your plate, please send a digital version (Excel or text file) of your sample names with associated well positions. 

Submission of Samples for Genotyping

Please let me know by email at least a day in advance that you will be submitting samples for genotyping!
Obtain a MicroAmp 96-well plate and septa compatible with the AB 3130xl (plates and septa are provided by MCF 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.  Please make sure that samples are well mixed.  Centrifuge your plate briefly to insure that the entire volume of each sample is at the bottom of the well with no bubbles.  Denature the samples for 3 minutes at 95°C and then immediately quick-chill them for 3 minutes on ice or in a benchtop chiller.  Please disable/do not use the 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.

Leave your plate(s) in the REFRIGERATOR in the Berry Center 320 and submit them using the online form.  Do not freeze samples containing labeled size markers!  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 (Excel or text file) of the sample names and associated well positions.  If no sample names are provided I will name the files with their well positions.  Please label the plate (not the septa) with your initials and a designation that I can associate with your sample file.

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

Project bulk discounts may be available. Please e-mail Terry at tmcclean@uwyo.edu for more information.

Use of MAC 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 MAC.

The MAC 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 MAC Service Core lab!  Refrigerator and/or freezer space is available.  NEW!  An equipment use/check out form is online for download and will be available in the Training/Prep Lab.  You must fill out a form for each visit to the Lab indicating which equipment you've used and your agreement to abide by MAC Policy.
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.

Citation language

Guidelines from the Association of Biomolecular Resources Facilities

Acknowledgement of facility contributions is expected in publications that include any data generated in the facility; for example, when fee-for-service is performed with no method development and minimum effort by Core personnel.
An example of an appropriate acknowledgement is "The authors wish to thank the Macromolecular Analysis Core at the University of Wyoming for analyzing samples".
Please inform us when relevant publications are accepted and forward us a copy for our records.  We may also post citation information on our website.  This information is vital to the continued support of the facility.

Please see the QA/QC policies at the UW MAC.