Submit forms to the operator listed below:
The Vision and Burch labs are running a ABI 3100-Avant Genetic Analyzer (a capillary electrophoresis machine with fluorescent detection) that is managed as part of the imaging core facility on the 4th floor of Wilson. The instrument can be used for DNA sequencing, SNP genotyping, and DNA fragment analysis.
The thermocycle and cleanup reactions must be performed in each investigator's lab. To reduce costs, we are purchasing reagents in bulk and selling them at cost through a freezer program. We are also compiling relatively simple, inexpensive and robust protocols for performing the necessary steps (all in 96-well plates).
The cost of most consumables associated with running the machine (e.g. capillaries, running buffer) are included in the lane fees (see below). Exceptions include the sequencing reaction mix (called Big Dye), fluorescent AFLP/STR primers, SNP genotyping kits (called SNPshot), and size standards for fragment analysis. These are either ordered individually (eg primers, plates) or sold at cost through a freezer program run out of the Burch lab. The reagents are stored in the Burch lab. They can be obtained only by requesting them, together with an account number, from the operator (the form is available for printout here). The reagents typically last about a year when stored properly. We expect sequencing reagents to cost a little less than a $1/rxn, assuming 1/8 volume reactions. Allow several days for Snapshot kits and size standard orders to be filled, as we do not keep an inventory of these in the freezer.
| Item | Vendor | Catalogue # | Cost | Note |
| 96 well plates | PGC scientific | 62-6049-02 (case of 25) 62-6049-03 (case of 100) | $52.50/25 $187.50/100 | Only samples submitted in these plates are accepted UNC account #: 72650 (UNC) |
| Sealing foil | MJ Research | MSF-1001-03 | $65/100 sheets | Refer to quote # VF052303E under name UNC-CH |
| Big Dye Sequencing Mix | Freezer program | See form | We are running 1/8 rxns which use 1 ul of Big Dye | |
| Sequencing Buffer | Freezer program or make your own | Supplied with BigDye | To make your own: 200 mM Tri-HCl pH 9 5 mM MgCl2 | |
| Snapshot Genotyping Mix | Freezer program | See form | Dilute rxns 1/10 to 1/20 | |
| Size standards (ROX/LIZ) | Contact Tony Perdue for ordering information | Choose based on dye set and fragment sizes | ||
| Formamide | Freezer program | See form | Only needed for fragments and SNPs One aliquot sufficient for one plate | |
| Fluorescently labelled primers | ABI | Inquire about UNC negotiated rate | Be sure to use the correct dye set |
For sequencing, we are using the Big Dye Terminator v3.1 chemistry.
For fragment analysis, the following table lists dye and filter set combinations currently in use. Others can be used, but the user must supply the spectral calibration reagents. The size standard that must be used with each filter set is shown in red. Note that there is a choice of short and long size standards for each dye set, the selection of which depends only on the lengths of the fragments being analyzed.
| Dye Set | Filter Set | Blue | Green | Yellow | Red | Orange |
| DS-30 | D | 6-FAM | HEX | NED | ROX | |
| DS-33 | G5 | 6-FAM | VIC | NED | PET | LIZ |
The ABI recommended protocols can be downloaded from here
Our own favorite protocols:
Tony Perdue is overseeing operation of the machine and managing the accounts but not running samples. One person other than Tony is designated to operate the machine on a day to day basis (a commitment of several hours per week). That person is listed below. The idea is to recruit a graduate student or postdoc from one of the labs using the machine to operate it for one semester in exchange for steeply discounted usage of the machine (please talk to Christina Burch or Todd Vision if you or someone in your lab may be interested!).
Accounts will be tabulated monthly, and the PI will recieve a statement indicating what charges have been made to each account for freezer program reagents and lane usage. Questions concerning these statements should be addressed to Tony Perdue.
There will be a lane (or well) charge for each sequencing reaction, or set of fragments. These charges help defray the cost of the machine itself and the consumables that are not sold through the freezer program (e.g. capillaries and polymer). The charges are reevaluated periodically for potential readjustment. Lanes are charged in sets of four (as the autosampler loads four capillaries at once), but additional blank wells from under-full plates are not charged.
The current charge is $5/lane, independent of what reaction is being run.
At the moment, we are running all samples (both sequencing and genotyping) on 50 cm capillaries. Alternative capillaries (which can offer greater resolution or speed, depending on your application) can be used for bulk orders (multiple plates) only. If you would like to use different capillaries, please talk to the operator.
If you are budgeting for a grant proposal, steep discounts may be possible in exchange for a contribution toward the service contract. Contact Christina Burch or Todd Vision to discuss special arrangements.
Contributors to the capital cost of the machine (you know who you are) are receiving dollar for dollar lane charge credits, and pay only the at-cost rate (currently estimated at $2.50) until their capital investment is recouped.
All samples must be submitted in a 96 well plate. For fragment analysis, samples are to be submitted frozen, already denatured in formamide and with the size marker included. See protocol. For sequencing, samples must be submitted as dried pellets.
Please leave the first four wells (A1-D1) empty so that controls can be introduced to the plate, one per capillary. The controls allow us to ensure that the machine is operating properly.
An account number for lane charges will be required at the time of submission (unless the fee is being waived for one of the reasons mentioned above). The plate submission form is available here. We currently only accept plates with at least 12 wells. This number may be adjusted upward as usage increases, and we may ask that users combine their samples onto a single plate when backlogs begin to accumulate.
For standard sequencing reactions, the machine can process about half a plate (48 lanes) per day. Samples will be processed on a first-come first-serve basis. For unusually short or long sequencing reactions, SNP reactions, or fragment analysis reactions, the running time may vary.
Sequence analysis: Most users will find the features they need in VectorNTI and Sequencher (available at a discount through Center for Bioinformatics site licenses) or through server applications like Phred/Phrap/Consed (available on Biolinux). Several shareware or freeware chromatogram viewers are available for both Mac and Windows (e.g. FinchTV).
Fragment/SNP/AFLP analysis: There is one copy of GeneMapper vers. 3.7 installed on the computer "mint" in the Vision lab (Coker 122). The login password is "basepair". Users are welcome to use mint in the lab and also to borrow the Genemapper User's Guide, which is kept on the shelf above mint.
It is also possible to remotely access and use Genemapper on mint. Windows users should open the Accessories->Communications->Remote Desktop Connection application from your Program Pop-up Menu. In the address field, type "mint.bio.unc.edu", or "152.2.15.147". This will open up a remote session on mint and temporarily displace your current desktop. Mac users need to download and install a free client. Once connected, you can login to mint using your regular Biology computer account, but you need to have been first granted access; contact Todd Vision if you need access and do not yet have it.
Unfortunately, only one user can be logged in to mint at a time, either remotely or locally. So it is very important to log out when you are not actively using the software. Be sure to explicitly log out of the session, rather than simply close the window, as the latter will not free up the computer for others to use.
Alternative fragment analysis software programs do exist. Some that we are aware of include Genographer (although the web site does not appear to working at the time of writing), STRand and GeneMarker.
Data is stored on Bioark//Avant/Out in individual user directories. Users are notified by email when their data becomes available. Data older than one month old will be removed by the operator, so please copy your data elsewhere and do not use the Avant server for long-term data storage!
For help with troubleshooting, these pages have examples of good and bad sequencing electropherograms and some hints for fixing specific problems:
The person to whom requests for use of the machine are submitted (for Summer 2005) is Monica Meng in Christina Burch's lab.
Starting mid-August, the operator will be Ashleigh Huggins from the Pukkila lab (email).