Molecular Biology & Proteomics Core Facilty Dartmouth College

DNA Sequencing Service General Information

Introduction

DNA sequencing is performed on the Applied Biosystems Model 3100, a capillary based electrophoresis system that uses fluorescent dye terminator chemistry. For details on the features of the instrument and chemistry, see the Applied BioSystems website. The Molecular Biology Core Facility has two 3100’s. Using our standard run, each is capable of sequencing 16 samples in 2.5 hours and up to 196 samples (about 120Kb) per 30 hour. run. There are two methods of submitting your sample for DNA sequencing. We highly recommend that you submit template and primer to the facility in the recommended amounts and have us do the chemistry. The advantage is that your samples are treated uniformly and processed with control samples, using more efficient methods, and it is cheaper. Alternatively, you may do the reactions and subsequent purification of the extension products yourself; please download these instructions.

Sample Submission

Sequencing orders are placed through the Core Server. Instructions for using the server are available here and in the download section of this website. Copies of your order with your sample(s) can be dropped off at any time at the laboratory in 241 Remsen, the Vail 200 Darkroom, the Borwell pick up site room 716E or Rubin 7. Data from samples submitted before 11:00 AM at any site will usually be reported to you the next day. If you perform the reactions and cleanup yourself, the samples will be accepted for next day service until 3:00 PM in 243 Remsen.

Data Distribution and Quality

When the sequencing is completed, you will receive your data by e-mail. Each sample will have two files associated with it. One file will show the electropherogram for your sample and the run and analysis conditions. You will also receive a file with a .seq extension. This file is a text file with raw sequence called by the computer. This file can be viewed using Microsoft Word. If this file is empty or contains only N's, your sample was unsuccessful. The ABI Prism 3100 DNA Sequencer operates under Windows NT therefore the data files are in Windows format and will need to be converted to be viewed on a Mac.

We like FinchTV for viewing electropherograms it is available for free from the Geospiza website. FinchTV operates on both Mac and PC platforms and no conversion is needed. Mac users can use EditView1.0.1 available from Applied BioSystems web site. With EditView1.0.1 a data file can be edited to correct problems with the computer-called sequence. If you are a PC user, you can download demo software from Chromas to view and edit your data. Please purchase the shareware version of Chromas.

Always look at the data file and be sure your sample had a strong enough signal, proper base spacing and that the appropriate base caller was used. If you have sufficient non-problematic template, a good primer, and clean extension products, it is not unusual to get 600-700 bases of unambiguous data from our standard run. Once you develop some proficiency at editing the computer-called sequence, you can often get another 50 bases. Our standard typically sequences to 97% accuracy at 700 bases, unedited. We cannot emphasize strongly enough that the quality of you sequence data depends on the purity and quantitation of your template and the quality of your primer.

Cost

The cost of a DNA sequence is $10.50 per sample when we perform the sequencing reaction and purification of extension products and $8.00 per sample for completed reactions. See the sample sheet for a complete price list.

Turnaround Time

Turnaround time is usually 24 hours or less for all reactions. However, this is highly dependent on the number of samples in the queue and the time of day the sample arrives at the laboratory. Generally, we start processing samples (template and primer submissions) for cycle sequencing at 11:00 AM. All completed reactions are loaded on the sequencers at 3:00 PM for the overnight run. On busy days, all samples may not be finished by the morning. The later your samples arrive at the lab, the later the data will be reported out. On busy days, the data for later arriving samples will be reported out during the following day as they finish.


				DNA Sequencing Service General Information Introduction DNA sequencing is performed on the Applied Biosystems Model 3100, a capillary based electrophoresis system that uses fluorescent dye terminator chemistry. For details on the features of the instrument and chemistry, see the Applied BioSystems website. The Molecular Biology Core Facility has two 3100’s. Using our standard run, each is capable of sequencing 16 samples in 2.5 hours and up to 196 samples (about 120Kb) per 30 hour. run. There are two methods of submitting your sample for DNA sequencing. We highly recommend that you submit template and primer to the facility in the recommended amounts and have us do the chemistry. The advantage is that your samples are treated uniformly and processed with control samples, using more efficient methods, and it is cheaper. Alternatively, you may do the reactions and subsequent purification of the extension products yourself; please download these instructions. Sample Submission Sequencing orders are placed through the Core Server. Instructions for using the server are available here and in the download section of this website. Copies of your order with your sample(s) can be dropped off at any time at the laboratory in 241 Remsen, the Vail 200 Darkroom, the Borwell pick up site room 716E or Rubin 7. Data from samples submitted before 11:00 AM at any site will usually be reported to you the next day. If you perform the reactions and cleanup yourself, the samples will be accepted for next day service until 3:00 PM in 243 Remsen. Data Distribution and Quality When the sequencing is completed, you will receive your data by e-mail. Each sample will have two files associated with it. One file will show the electropherogram for your sample and the run and analysis conditions. You will also receive a file with a .seq extension. This file is a text file with raw sequence called by the computer. This file can be viewed using Microsoft Word. If this file is empty or contains only N's, your sample was unsuccessful. The ABI Prism 3100 DNA Sequencer operates under Windows NT therefore the data files are in Windows format and will need to be converted to be viewed on a Mac. We like FinchTV for viewing electropherograms it is available for free from the Geospiza website. FinchTV operates on both Mac and PC platforms and no conversion is needed. Mac users can use EditView1.0.1 available from Applied BioSystems web site. With EditView1.0.1 a data file can be edited to correct problems with the computer-called sequence. If you are a PC user, you can download demo software from Chromas to view and edit your data. Please purchase the shareware version of Chromas. Always look at the data file and be sure your sample had a strong enough signal, proper base spacing and that the appropriate base caller was used. If you have sufficient non-problematic template, a good primer, and clean extension products, it is not unusual to get 600-700 bases of unambiguous data from our standard run. Once you develop some proficiency at editing the computer-called sequence, you can often get another 50 bases. Our standard typically sequences to 97% accuracy at 700 bases, unedited. We cannot emphasize strongly enough that the quality of you sequence data depends on the purity and quantitation of your template and the quality of your primer. Cost The cost of a DNA sequence is $10.50 per sample when we perform the sequencing reaction and purification of extension products and $8.00 per sample for completed reactions. See the sample sheet for a complete price list. Turnaround Time Turnaround time is usually 24 hours or less for all reactions. However, this is highly dependent on the number of samples in the queue and the time of day the sample arrives at the laboratory. Generally, we start processing samples (template and primer submissions) for cycle sequencing at 11:00 AM. All completed reactions are loaded on the sequencers at 3:00 PM for the overnight run. On busy days, all samples may not be finished by the morning. The later your samples arrive at the lab, the later the data will be reported out. On busy days, the data for later arriving samples will be reported out during the following day as they finish.


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