Automated DNA Sequencing
SUMMARY OF SERVICES
Sequencing
The BMGC sequencing and genotyping facility utilizes capillary electrophoretic DNA analyzers for quick turnaround on small and high-throughput projects. Three 16 capillary ABI 3130xl and one 96 capillary ABI 3730xl use ABI BigDye version 3.1 Terminator chemistry.
PRICES
Individual tube - University CUFS number
Plate submission - University CUFS number
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$3.50 per sample for plasmid or PCR products in 96-well plate (run on 3730xl)
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$7.50 for BAC (full-reaction) samples for 96-well plate
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$6.00 for BAC (half-reaction) samples in 96-well plate
Individual tube - Affiliated company or Educational purchase order
Plate submission - Affiliated company or Educational purchase order
Individual tube - External user purchase order
Plate submission - External user purchase order
HOW TO ORDER
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For an individual sample, prepare and submit sample with appropriate template/primer concentrations in a 0.5 mL tube along with the DNA Sequencing Request Form. To place an online order go to https://www.ahcs.umn.edu/bmgc/index.cfm. Recommended template and primer concentrations (concentrations may need to be adjusted for each researcher) are as follows:
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In a 0.5 mL tube submit the following:
optimized template (see list at right)
3.2 pmole of primer
Water to a final volume of 12µl
For 96-well plate quantities please inquire.
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PCR size Quantity
100-200 bp 1-3 ng
200-500 bp 3-10 ng
500-1000 bp 5-20 ng
1000-2000 bp 5-20 ng
>2000 bp 20-50 ng
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Single-stranded DNA
25-50 ng
Double-stranded DNA
150-300 ng
Cosmid, BAC, Bacterial DNA**
0.5-3 µg
**additional charge applies
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For plate submission, prepare and submit plate with appropriate template/primer concentrations with the DNA Sequencing Request Form. To place an online order go to https://www.ahcs.umn.edu/bmgc/index.cfm. Recommended template concentration is half the amount used for optimized samples submitted in individual tubes. Primer concentration is 3.2 pmole per well. The final volume per well is 6 uL. Samples are not reprocessed and no hard copies are included. Plates need to fit our thermal cyclers and can be purchased through the facility. A pack of 10 plates can be purchased through www.appliedbiosystems.com, part numbers 4346907 or N801-0560, or 50 Corning plates #3753 can be purchased through UStores order number 07201216. Cover plates using plate tape available through UStores part number CX18443 (Corning #6524 pack of 100). Please contact a staff member before submitting a plate for the first time.
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Minimum number of samples needed for plate submission is 48.
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If partial plate is submitted, samples need to be loaded from A1-H1, A2-H2, A3-H3, etc.
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Empty wells must be guaranteed to be clean and empty. Please note this in the addional comments on your DNA sequencing request form.
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It is recommended to leave A1 and H12 open for controls.
General guidelines for all samples:
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Primers used for sequencing are best between 18-22 bases in length with a predicted Tm between 50-55ºC. G-C content of 40-60% is desirable. Avoid primers with strong self-dimers or hairpins. Also, avoid primers which are >90% homologous to other regions within your sample. A BLAST search will alert you to this problem.
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The best sequencing data is found 80-150 bases away from the primer. It is difficult to get good basecalling less than 50 bp away from the primer.
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There is no extra charge for samples with 10% DMSO (check DMSO box).
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BAC samples require more template (try starting with 1 µg of template).
What paperwork needs to be completed?
Where can samples be dropped-off?
We have 2 drop-off locations
Sending samples – mailing address
Sending samples – shipping address
Where can results be obtained?
Results are in two different formats: text (.seq files) and
trace (.ab1 files).
Placement of results: BMGC Data Management System (posted for 30 days), FTP SITE, or HARD DRIVE
What is the turnaround time?
We have a 72-hour turnaround time. If you have not received an email message before the 72-hour turnaround time, please contact any staff member at 612-625-7736.
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