Sequencing & Genotyping Facility
Eberhart Building Room 226
The UNCG Sequencing and Genotyping facility is located in Eberhart 226. The facility provides access to automated sequencing equipment and data processing resources. Access is on a fee for use basis.
May 26, 2006 — All users please make note that capillary G1 has been damaged. Avoid loading this well on your sample plates.
August 16, 2006 — All users please check the quality of the skirted plates you are using. If there is any type of malformation do not use the plate in the sequencer. A warped plate may damage the instrument. It is suggested to use the following plates in the Megabace 500: Product No. MSP-9601 available from Bio-Rad. These plates are the same specifications as the ones previously available from MJ Research.
MegaBACE 500 DNA and Genotyping Analysis System
Specifications and supported uses:
The MegaBACE 500 is a 48-capillary dual-laser DNA sequencing and analysis system manufactured by GE Healthcare (formerly Amersham Biosciences), using a linear polyacrylamide (LPA) matrix for resolving samples. Supported uses are:
- DNA sequencing
- Marker genotyping (SSR, TRFLP, potentially AFLP)
- NP genotyping
DNA sequencing specifications:
- Current filter set accommodates GE Healthcare ET sequencing chemistry.
- Standard sequencing reaction cleanup is done with ethanol precipitation; Sephadex column cleanup is another option.
- Sequence analysis is performed with MegaBACE Sequence Analyzer, supplemented with MegaBACE ScoreCard and Sequencing Diagnostics.
- Chromatograms (.abd format) are ABI-compatible.
Marker genotyping specifications:
- Filter set accommodates HEX, FAM, and NED/TAMRA dyes for primer labels, which can be ordered from preferred oligo supplier.
- ET-ROX size standards with various size ranges must be purchased from GE Healthcare.
- PCR cleanup uses dialysis into Millipore filter plates. [This may change due to changes in available product specifications.]
- Data analysis is performed with MegaBACE Genetic Profiler or Fragment Profiler.
SNP genotyping specifications:
- Uses same filters as sequencing.
- Uses GE Healthcare SNuPe chemistry.
- Data analysis is performed with MegaBACE SNP Profiler and SNP Marker Editor.
B. Use Policy:
All first-time users MUST receive training and hands-on supervision from the Facility Manager or (with prior Manager approval) from an experienced user.
All users must sign up in advance for available time slots.
- Allow a minimum of 2 hours for sequencing and genotyping runs, including matrix fill, prerun and loading time.
- Users more than 15 minutes late for starting a run must forgo their time slot.
- If plans change, users must remove their names from the sign-up as soon as possible.
- Users who decide to cancel a run the day of their scheduled use or the day before their scheduled use MUST also ensure that capillaries are stored appropriately (see item 4 below).
- Sign-up is on a first-come, first-served basis.
All users must fill out the user log for runs and for other operations performed (e.g. store capillaries, flush and dry).
Prior to starting a run, users are responsible for ensuring that the correct filters (sequencing or genotyping) are installed for their planned use, and for selecting the correct use in the Instrument Control Manager.
If no users are signed up for runs later in the same day, you must ensure that capillaries are properly stored or flushed.
- If a user is signed up for the following day, check “sleep after this run” and set the time at 16 hours. (This can be done any time before the run is complete.)
- If the next user is signed up for 2 or 3 days later, or if the run is already completed, you MUST return after your run, and run the Store Capillaries program with the sleep time set at 72 hours. (This will take about 5 minutes to do.)
- If the next user is signed up for more than 3 days later, you MUST return after your run, and run the Flush and Dry Capillaries program. (This will take about 25 minutes to do).
- Failure to follow the above practices will probably damage the capillaries, and require purchase and installation of a new set of capillaries (approximately $2200). If this happens, the negligent user will be responsible for the cost of new capillaries.
If any problems occur before or during a run, and you are not COMPLETELY sure how to handle them, you must do the following:
- Contact the Facility Manager or an experienced user who can solve the problem.
- If no one who can help is available, call GE Healthcare Technical Support at 1-800-526-3593. (They are usually very helpful and normally can talk you through the problem to solve it.)
Facility Resource Fees and Billing:
All outside users (UNCG users outside Biology Department and non-UNCG users) must make arrangements for billing and payment prior to using the facility.
The fees listed in the schedule below will be billed to user accounts quarterly. The number of runs will be determined from the user log and the number of data folders created, and any discrepancies in these records will be resolved by the Facility Manager.
Billing for fixed costs per run (amount in item A in the schedule below) MAY be waived for UNCG Biology Department users on a quarterly basis at the Department’s discretion, depending on available funds. Fixed costs include pro-rated cost of capillary replacement and expected frequency of service calls.
Arrangements for UNCG purchase of reagents and consumables (items B-D in the schedule below) must be made ahead of time with the Facility Manager in order to ensure availability of reagents and verify availability of funds for their purchase.
Per-run costs (item A or B in the schedule below) will apply whether or not a complete set of 48 samples is run, and whether or not the run yields useful data.
Per-reaction costs (items C and D in the schedule) are based on UNCG’s purchase cost, and will be charged whether or not reactions are successful. Users are strongly encouraged to study sequencing and genotyping protocols and MegaBACE MegaManual prior to beginning a project in order to increase the likelihood of successful results.
Minor users (i.e. those who will seldom have full runs of 48 reactions at one time) are encouraged to coordinate their use with other users to fill out a full plate and purchase reagents.
The following schedule will be updated as needed, based on the most recent prices and costs. Any changes will become effective at the start of the next billing cycle.
|A. MegaBACE run, user provides LPA matrix/buffer||$31.00||run|
|B. MegaBACE run, Biology Dept. provides LPA matrix/buffer||$57.00||run|
|C. Sequencing reactions: Biology Dept. provides reagents:
• 10 µl reactions using dilution buffer
• 20 µl reactions using dilution buffer
• 10 µl reactions without dilution buffer
• 20 µl reactions without dilution buffer
|D. Genotyping reactions: Biology Dept. supplies size standards, user provides other reagents and supplies||$ .33||rxn.|
If you are an internal user interested in booking time please contact the facility manager to do so.
Current users may log in to Calcium here. After log in you will be able to view facility calendars and book available time slots.
View MegaBACE 500 availability here.
David L. Remington, Ph.D | Phone: 336-334-4967
Technical Staff / Facility Manager