DNA Sequencing Core

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The DNA Sequencing Core at Center for Gene Therapy, Tulane University Health Sciences Center operates two ABI Prism 3100 sequencers. These sequencing reactions are performed by using ABI BigDye^TM Terminator chemistry (Applied Biosystems).

These sequencers use the same capillary technology that was widely publicized during the last two years in the rapid analysis of human genomic DNA (ABI Prism 3700). Instead of 96-capillaries, this new model (released in July, 2000) uses a 16-capillary loading system to better serve mid-size sequencing facilities. Compared to the gel-based sequencing systems, the advantages of the capillary sequencer include a lower error rate (sample loading automated, gel/polymer issues virtually non-existent), higher through-put and reduced cost of operation. However, of course, the reagent cost, instrument service contracts and labor cost still exist, and therefore, we do have to charge for the sequencing analysis.

The charge per reaction is $15. If the reactions are brought in 96 reaction- sets in 96-well format PCR-plate, the charge per reaction is $10.

With the best samples we can achieve >750-bp read-lengths but the error rate starts to get significantly higher after ~650-bp. With these two instruments we can process about 320-440 samples a day depending in the read-length. Currently we run about 100 samples a day. Thus, we can easily commit to 200 additional reactions a day. If necessary, there is also an option to start operating the sequencers during the weekend for additional 600 or so samples a week.

We have 4 years of experience in running a middle-throughput DNA sequencing core and are committed to helping in-house researchers in all aspects of the sequencing (primer design, template purification, data analysis), not only in running the samples.

Primer walking- projects can be performed either relying on your primers or we can manage the primer design. If we are using your primers, the cost per reaction is $15. If you want us to walk the sequence, the cost is $0.15/base pair (includes the primer design, primer ordering, the sequencing reaction, the sequence assembly).

For any questions please call Justin (504-988-7063) or Mimi (504-988-7706). We are located in Tulane Medical School campus, Tidewater Building, room 2140. Between 8.15am-5pm you can just walk in and someone will be in the lab 98% of the time to take the samples.

SAMPLE PREPARATION

All samples must be submitted with a sequencing requisition form. An IT must be submitted at the beginning of each month.

Click here for sequencing form.

PCR Product

Remove the remaining primers and dNTPs from the PCR reaction with your favorite purification method. Prepare the reactions in a 0.2 ml PCR reaction tube using the following guidelines:

Plasmid

Purify the plasmid DNA with your favorite purification method. The cleanliness of the sample is paramount in plasmid sequencing.  Prepare the reactions in a 0.2 ml PCR reaction tube using the following guidelines: