DNA Sequencing
- General Information
- How To Order
- Sample Submission in detail
- Troubleshooting Guide
- Viewing Your Sequencing Results
General Information
We use the Applied Biosystems Model 3730 capillary DNA sequencer with Big Dye Terminator Cycle Sequencing Kit. Maximum read lengths are approximately 1000 bases. The sequence is readable about 30–50 bases downstream of the primer sequence. We anneal at 50°C and extend at 60°C so make your primer melting point (Tm) ca. 55°C. You may want to do a Google search for “Oligo Molecular Weight Calculator” to find web pages that will calculate Tm quickly.
Primer Considerations
- High Purity
- Appropriate concentration
- No mismatches
- No alternative hybridization sites in template (false priming)
- No palindromic sequence present, particularly at the 3’ end of primer
- Appropriate length to give Tm of ~55-60°C. (generally 20-24 bases)
- GC "clamp" on the 3' end
- Desirable [GC] = ~50-55%
- Avoid strings of four or more of same the base if possible
- Avoid low Tm (i.e. 40-45°C.) If Tm is low, make the primer longer.
Template/Primer Quantity Table
Recommended template and primer concentrations in a 12ul sample. If your sample and primer combined is less than 12ul add deioinzed water to bring it up to 12ul. Template/Primer should be submitted in THE SAME tube.
| Template | Quantity |
|---|---|
| PCR Products: | |
| 100-200 bp | 1.5-9 ng |
| 200-500 bp | 3-30 ng |
| 500-1000 bp | 6-60 ng |
| 1000-2000 bp | 15-120 ng |
| >2000 bp | 30-150 ng |
| Single-stranded DNA | 30-150 ng |
| Double-stranded DNA | 400-900 ng |
| Primer | 10 pmoles |
| For the following templates submit sample and primer in SEPARATE tubes: | |
| Template | Quantity |
| Large DNA (>30,000bp) BACs, PACs, YACs, cosmids, fosmids | 0.6-3.0 ug at 150ng/ul |
| Bacterial Genomic DNA | 3-9 ug at 150ng/ul |
| Primer | 5ul at 10pmol/ul (10uM) |
Template Preparation for Successful Automated DNA Sequencing
How to Order
We use a laboratory information management system (dnaLIMS) to handle orders and distribute data. Templates are mixed with primers and submitted in 12 ul as described in the Template Primer Quantity Table section. Also see the Sample Submission in Detail section for additional information. Requests are submitted to:
First time users must create an account before logging into the LIMS. Once logged in, follow the instructions and call if you have any problems. Your account number must be exactly seven digits with no spaces, dashes etc.
The LIMS asks if the template is “difficult to sequence”. We will use extra polymerase, blend polymerases and / or alter the thermocycling conditions if you select “difficult template” and also indicate the nature of the difficulty (e.g. hairpin or GC rich) next to the samples in question when you fill out the web request form.
Print out 2 copies of your request—one for yourself and one to submit to the lab. Drop off your samples and request forms in the refrigerator at the front of the lab. The file name of your data will consist of your sample name and primer. For example, if your sample name is AB123 and the primer is M13F the electropherogram file name will be AB123-M13F.ab1. Submit samples in 8-strip PCR tubes or 96-well plates. Use only ABI (PN: N8010560) plates which you may obtain from us. Place strips and tubes into 50cc tubes and write the assigned “Order Number” on the tube or plate.
Sample Submission in Detail
If you have any questions about sample preparation for DNA sequencing that are not addressed on this page we suggest that you download the Applied Biosystems DNA Sequencing Chemistry Guide and/or the Qiagen publication: “The QIAGEN Guide to Template Purification and DNA Sequencing, 2nd edition” from their web sites. We will also email these documents to you upon request.
How to Prepare samples for DNA sequencing
Individual tubes
Template and primer should be diluted to 12 ul with water. Submit samples in 8 strip PCR tubes with caps (ie.VWR/MIT Stockroom PN 20170-004) Label tubes 1,2,3 etc. DO NOT try and squeeze your sample name on the tubes. It makes it very difficult to read and can result in a delay if we need to contact you because we cannot read the tubes. Submitting samples in eppendorf vials takes longer to set up so PLEASE USE STRIPS. Group individual tubes/strips into 50 ml Falcon tubes. (we have recycled 50 ml tubes in the lab near the refrigerator) Label with NAME and ORDER NUMBER. Place in refrigerator at front of lab and leave forms in the plastic pocket.
96 well plate(≥75 samples)
ONLY use ABI plates (PN: N8010560) which can be obtained from us. Scale down the template and primer requirements by 50% and dilute to 6 ul with water.
To receive the discounted price you MUST follow these instructions for submitting directly in 96 well plates:
Contact the lab and we will supply you with the correct plate to use. If samples are submitted in plates other than those specified, we will have to transfer the samples into the correct plates, therefore, the price will not be discounted.
Load the plate vertically NOT horizontally. The instrument picks up vertical columns at one time. When the data is posted it is arranged by vertical wells, A1-H1, B2-H2, C3-H3 etc. If you load horizontally your data will not post in the same order as you see on the request sheet. We cannot change the order in which data posts.
You MUST use only 6ul if directly loading a well plate. If you use 12ul we will have to transfer 6ul of all the samples to another plate defeating the whole purpose of submitting it in the plate, and therefore, the price will not be discounted.
Leave A1 and/or H12 empty for our standard. It is preferable to have a standard in one odd well (A1) and one even well (H12). A single standard is OK if you have 95 samples. We can run a plate of 96 samples but please be aware there will be no control sample to verify instrument performance.
Please Note: An order of fewer than 75 samples will not be eligible for a discounted price. If you have fewer than 75 samples it is preferable to use the 8 strip tubes.
Troubleshooting
Viewing the Sequencing Results
You will be notified by email when the results are ready. Inspect each electropherogram for errors in base calling. Occasionally, there are miscalls that are obvious to the eye but difficult for a computer to recognize. As a rule, when you reach the point where there is one N per 20 bases, the sequence is not usable.
You can view and print the electropherogram (sequencing traces) and text output along with a quality score. Files can be downloaded to your computer. For rapid turnaround, on weekends we may send out raw data without editing. If subsequent editing was needed you will receive a new mailing. Turnaround time is typically 48 hours.
The following web sites offer free programs for viewing electropherograms (.ab1 files):
| Software | Operating System |
|---|---|
| 4 PEAKS | Mac OS X |
| Sequencer Scanner | Windows |
| Sequencher | Mac OS 9 and below |
| Chromas | Windows |
| Codon Code Aligner | Mac, Windows and Unix |
Pay software to view electropherogram outputs
Your department may have a site license for DNA alignment, searching and manipulation programs such as DNASTAR, Sequencher etc.
