DNA Sequencing

Go to the UT DNA Sequencing Facility website. This page explains the pricing for various orders and the methods by which samples should be prepared prior to sequencing.

PCR Products

This method pertains to PCR products and gel extracted DNA. Before beginning the sequencing preparation, make sure the samples have been cleaned and had their concentrations measured. If there are 24 or more samples to be sequenced, use a 96 well plate for preparation; if there are fewer than 24, use 1.5 ml eppendorf tubes. For PCR products, the final volume of the reaction should be 12 μl (if including 1 μl of primer) or 11 μl (primers to be added by the DSF). The remaining volume should consist of template (coming from the PCR product) and water (sterile).

The amount of template needed for the reaction depends on the predicted length of the product, in that 2-5ng of template are needed for every 100bp of product. For example, if a product is expected to be 2500bp in length, the sample should contain 40-100ng of DNA. Calculations are necessary to determine what volume of cleaned PCR product is necessary, taking the concentration of the DNA. For example, using the example given above, 1-1.5 μl from a 60 ng/μl product should be appropriate. Continuing this example, the final volumes for this sample (assuming the inclusion of 1 μl of primer) might be 1.5 μl template, 1 μl primer and 9.5 μl sterile water.

Note: When using intermediate primers, make sure the correct product length is used for calculations. If a 3000bp PCR product uses an intermediate primer about 1800bp before the end of the sequence, that the sample containing this primer has an expected product length of 1200bp, not 3000bp.

When entering samples into a 96 well plate, go down a column, then over a row. For example, A1, B1, C1, ..., H1, A2, etc. Cover the well plate with aluminum foil and write the order number on the foil so that it is easily visible.

Placing the Order

To place an order for a sequencing reaction, first make an account or directly place an order and fill in the necessary fields. Make sure to select Jeffrey Barrick (Department: Biochemistry) from the list instead of manually entering a PI.

Once logged in, select Enter Individual DNA Sequencing Requests, enter the number of samples and select a service based on the descriptions given. Fill in a grant number (either startup or NIH - materials and supplies) and select Qiagen for DNA prep kit, although Zymo kits are the ones used in the lab.

When naming the template, DNA type and Primer, know that these names are only for personal interpretation and don't need to be the "official" names of the templates or primers. Note that primers are asked for in terms of picomoles, which will be 10 if 1 μl of 10μM primer was used. Enter the lengths of the products and the concentration in the sample (this will be mass of template in sample divided by 12 μl). Once all the data is filled in, submit the form and make note of the order number.

Make sure the order number is on the sample(s) and take it to MBB 1.426 and place it in the appropriate part of the "to be sequenced' cabinet.

-- Main.AurkoDasgupta - 29 Jun 2011

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Topic revision: r1 - 29 Jun 2011 - 18:36:51 - Main.AurkoDasgupta
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