Instructions: MDIBL Marine DNA Sequencing Facility
Our sequence analyses are performed on two 16 capillary ABI 3130xl model DNA Sequencers. The sequencing reactions utilize a 3'-fluorescently-labeled dideoxynucleotide triphosphate mixture (BigDye Terminator version 3.1) and Taq FS DNA polymerase in a thermal cycling protocol. A good quality DNA template can yield an average read length of 800 base pairs with exceptional accuracy. These instruments are also utilized for microsatellite and fragment analysis applications, and may be used with ROX, FAM, NED and HEX dyes.
Prices:
Sanger Sequencing
MDIBL/INBRE Investigators: $9/sequence
48-96 samples in 96-well plate: $6/sequence
External Investigators: $11/sequence
48-96 samples in 96-well plate: $8/sequence
Fragment Analysis
MDIBL/INBRE Investigators: $12 per run (one run = 1 to 16 samples)
$72 for a full 96 well plate
External Investigators: $14 per run (one run = 1 to 16 samples)
$84 for a full 96 well plate
Important Preparation Guidelines:
Template
Successful DNA sequencing results are highly dependent upon the quality of the DNA template supplied by the user. Careful quantification of your DNA is crucial.
- Concentration of template should be based upon OD260 results.
- Verify the concentration and check for overall quality by gel electrophoresis.
- Provide the template in sterile distilled water (preferred) or 10mM Tris-HCl, pH 8.0, but not TE buffer, as EDTA may interfere with the sequencing reaction.
Primer
Consider the following guidelines when designing primers for use in sequencing:
- 20-30 nucleotides in length
- Avoid runs of an identical nucleotide, particularly four or more Gs.
- Keep the G-C content in the range of 40-70%; the closer to 50%, the better
- A melting temperature of at least 50˚C
- Avoid primers that can hybridize to form dimers
- Primers ideally should be located 30-40 bases upstream of region of interest
Sample Submissions:
Provide primer and template premixed according to the following guidelines. The recipes are sufficient for two sequencing reactions, so if a technical failure occurs, we will not have to contact you for additional sample material.
For DS plasmid DNA:
500-600 ng ds plasmid DNA template
2 µL 4µM primer
+ x µLsterile water
24 µL total volume
For PCR Products:
Mix as above, except use 10 ng template for every 100 bases of PCR fragment length, up to 80 ng.
For Cosmid, BACs and Phage Lambda DNA:
Please submit 2-3 µg DNA template and provide 5 µL of 100nM primer in a separate tube.
For microsatellites/fragment analysis:
Please dilute your PCR reactions and size standard in HiDi Formamide, for a total volume of 10 to 15 µL per sample.
Sending samples to be sequenced:
If the DNA samples are delivered overnight, they may be sent at room temperature. Be sure to pack the tubes with care so the caps do not pop open en route, and the tubes do not break. Please use the following address for your shipments:
Christine Smith
MDIBL
Old Bar Harbor Rd., PO Box 35
Salisbury Cove, ME 04672
