The DNA Core accommodates both single sample users and high-throughput projects. The facility utilizes a 3730xl 96-capillary DNA Analyzer with Applied Biosystems Big Dye Terminator cycle sequencing chemistry.
Read lengths up to 1100 bases per reaction are routine, providing the template is of high quality. Turn around time for submissions is < 24 hours.
Staff are available for consultation or to review data. The DNACF Troubleshooting Guide provides examples of common sequencing issues and recommendations.
Fees are listed on the DNACF Fee List.
Submission Guidelines for Single Samples
- Sequence orders are placed on-line using the DNA Core's dnaLIMS system. Instructions on the use of the dnaLIMS system, and creation of an account, contact the DNA Core.
- All samples are to be submitted in a 1.5ml tube. The rack used by our Tecan robotic system restricts us to accepting only tubes with specific dimensions. The DNACF currently uses Fisher Scientific (Cat. #05408129)1.5ml MCT Graduated tubes for sample submissions. The Fisher Scientific tubes may be purchased from the Enzyme Freezer Program in a 500 tube per bag quantity.
- Total volume of the template + primer mix in the 1.5 ml tube is to be 16 ul.
- Recommended total amounts in the 16ul volume for DNA templates and primer:
- Each order receives an order number and each sample receives a requisition number. Label tubes with the last three digits of the requisition number on the top of the tube.
- Place samples in a plastic bag with the LIMS order number and submitting individual's name clearly written on the outside of the bag. (Plastic bags and permanent marker are provided on a cart outside of room 215).
- Place samples in the white refrigeration unit located in the hallway directly across from room 211. A box for samples will be on the left side of the unit on the second shelf from the top.
|DNA total mass
|Plasmid DNA(dsDNA)||< 5 kb||500 - 750 ng||20 pmoles|
| ||5 - 10 kb||750 - 1000 ng||20 pmoles|
| ||> 10 kb||< 1200 ng||20 pmoles|
|Single Stranded DNA)|| ||275 - 500 ng||20 pmoles|
|Purified PCR Product||< 500 bp||20 -50 ng||20 pmoles|
| ||500 - 1000 bp||50 -100 ng||20 pmoles|
| ||1000 - 2000 bp||100 -250 ng||20 pmoles|
| ||2000 - 5000 bp||250 - 500 ng||20 pmoles|
| ||5000 bp - 10 kb||750 - 1000 ng||20 pmoles|
| ||2000 ng||20 pmoles|
For example: Order #15759 has two sample; Requisition #289573 and Requisition #289574. Each tube is labeled with the last three digits of the requisition #.
Considerations for 96-well Plate Submission:
- Samples are to be submitted in a 96-well plate obtained from the DNA Core. Plates containing less than 96 samples should be loaded in a vertical fashion (A1, B1, C1, D1, E1, F1, G1, H1, A2, B2, etc.)
- Total volume of the template + primer mix in each well of the plate is to be 6 ul.
- Add 5 - 10 pmol of a single primer to each reaction.
- The 3730 is sensitive to salts and other contaminants. Care in template preparation is essential to quality data and instrument maintenance.