Sequencing
Customized and High-Throughput Sequencing Services
Custom DNA sequencing is provided utilizing the Beckman CEQ™ 8000 Genetic Analysis System and the GE Healthcare MegaBACE™ 1000 DNA Analysis System.
There are several types of sequencing services offered including "quicklook" single read reactions, direct sequencing of PCR products, single strand gene walking, double strand gene walking, and primer design and gene walking. At the conclusion of the project customer will receive a text file of DNA sequence (via e-mail), a copy of the electrophoretogram and assembled sequence where required. BioServe will provide universal primers for sequencing at no cost.
For each sequencing reaction, customers should send 2 µg of template DNA and at least 20 pmoles of primer in a final volume of 20 µl. If you are sequencing with a universal primer or if BioServe has your sequencing primer, the tube should only contain 2 µg of template in a total volume of 19 µl. BioServe will add 1 µl (containing 20 pmol) of primer.
A control sequencing reaction is performed with each set of customer samples. The template is PGEM with a M13 primer. The quality of the control sequencing chromatogram (available upon request at no cost) is used to evaluate the quality of the overall reactions. If a sequencing reaction is of poor quality, but the quality of the control sequence is good, the reactions will be billed.
Once the sequencing reactions are complete, hard copies of the sequence chromatograms, text versions of the sequence, and a electronic text copy of the sequence can be provided. There is an extra charge for delivery of hard copies of the sequence chromatogram if sent by overnight courier.
Currently, we are not preparing sequencing templates. If you choose to prepare your own template, we recommend using a commercially available kit such as Qiagen MINI-Prep or Promega Wizard mini-prep system. The purity of the DNA template directly influences the quality of your DNA sequences.
Quick Look Sequencing
QuickLook sequencing is performed to determine the presence of an insert, the orientation of a fragment or to confirm the reading frame. Up to 700 bases can be obtained by this service. This usually requires a single sequencing reaction. The turnaround time for this service typically is 2-3 days. The customer receives a hard copy of the sequence and an electrophoretogram.
When requesting this service please provide the following:
- Minimum of 2 µg of CsCl or Qiagen purified DNA
- Vector type and size
- Cloned fragment size
- Sequencing primers (if not universal)
Direct Sequencing of PCR Products
PCR fragments submitted for sequencing should each be a single amplified fragment which has been analyzed on a PCR check gel (gel picture is requested). For best results with PCR fragments, we routinely use Clontech columns for purification. Results will be submitted with a hard copy of the sequence and electrophoretogram. Turnaround time for this service is usually 4-5 days.
When requesting this service, please provide the following:
- OPC® purified primers for sequencing
- 500 ng of amplified product per sequencing reaction.
- Gel picture of PCR product
Gene Walk
Gene walk sequencing projects are tailored to fit the individual needs of each customer. This service can be performed on either one strand or both strands of DNA. Gene walks provide contiguous sequence data for the region of interest. The turnaround time for these projects varies depending on the size of the fragment to be sequenced. BioServe utilizes the Beckman CEQ™ 8000 Genetic Analysis System sequencer or GE Healtcare's MegaBace 1000M and primer walking strategy to sequence the DNA fragments. At the end of the project, BioServe provides the customer with an assembled contigious sequence, a list of primers used, sequence generated from each primer and electrophoretograms.
When requesting this service, please provide the following:
- 100 µg of CsCl or Qiagen purified DNA
- Vector type and size
- Cloned fragment size
- Initial sequencing primers (if not universal)
