DNA Sequencing - FAQ
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Q. Who should I contact if I have questions about DNA sequencing? A. For questions about sequencing please call Dr. Mei Han at (205) 975-2012 or see our Contact page .
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| Q. How much do you charge per sequencing reaction? A. We charge $6 per sequencing reaction. A sequencing reaction is defined as one template + one primer. |
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Q. Do you give any discounts for large volumes of sequencing? A. Yes, we charge $432/95-well plate (one well left empty for control reaction.) We request that you contact Dr. Mei Han at (205) 975-2012 in the Core Facility prior to bringing a 95-well plate so that she can give you further instructions. |
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Q. How do I electronically submit my sample for sequencing? A. This can be done on the Sequencing section of our web page. First you need to create a user profile. After creating a user profile, you will be able to submit samples online to be sequenced. For directions on how to create a user profile, please click here. If you have problems entering a user profile, please contact Dr. Mei Han at 975-2012. |
Q. How much template and primer do I need to submit for sequencing? A. We request that you bring 10 ul of template and 10 ul of primer. |
Q. Is there a particular concentration of template/primer at which I should submit my samples in order to get optimal results? A. Yes. Please refer to the table for the appropriate concentrations of template and primer to submit to get optimal sequencing results. |
Q. How do I figure out the concentration of my oligo? A. Your oligo supplier includes an oligo specification sheet when they ship your oligo. The information on the spec sheet includes: oligo name, oligo sequence, # bases, molecular weight (MW), nmols/OD260, g/OD260, GC content, and Tm (50mM NaCl). Also the amount of oligo synthesized is reported in terms of OD260, nanomoles and mg. Using the the nanomoles of oligo synthesized and the desired concentration of the primer, you can calculate the amount of TE to add to make a 100 uM stock solution. NOTE: Always resuspend oligo stocks in TE. (mols of primer/ desired concentration of primer) = volume of TE to resuspend primer (87.16 nmols / 100 uM) = 871.6 ul (87.16 x 10-9 mols / 100 x 10-6 mols/L) = 0.8716 x 10-3 L = 871.6 ul of TE Now that you have your 100 M oligo stock solution, you need to dilute a portion of it to the correct primer concentration for sequencing your plasmid, 1.6 M. uM is equivalent to pmol/ L. 1 pmol/ ul = 1 x 10-12mols /1 x 10-6 ul = 1 x 10-6 mols/L= 1 uM. To make 100 ul of oligo working stock at 1.6 uM, follow this formula: (final desired concentration X final volume)/ stock concentration =volume of stock to use (1.6 uM X 100 ul)/ 100 uM = 1.6 ul = volume of stock to use 98.4 ul = volume of water 100 ul = total volume of oligo working stock. NOTE: Always dilute oligos in water to make working stocks. The number of mols of primer synthesized can be calculated by dividing amount of oligo in grams by the molecular weight of the oligo. 0.58 mg / 6658.4g/mol = mols of oligo 0.58 x 10-3 g / 6658.4 g/mol = 8.71 x 10-8 mols =87.1 x 10-9 mols = 87.1 nmols oligo |
Q. I am submitting several samples that will be sequenced with the same primer. Do I need to send 10 ul of primer in individual tubes for each template? A. No, there is no need to send 10 ul of primer in individual tubes for each template. Instead, please send one tube containing the following volume: [# Templates X (2 ul of primer/template)] X 1.5 = volume of primer to submit |
Q. I am submitting a single template that will be sequenced with several primers. Do I need to send 10 ul of template in individual tubes for each primer? A. No, there is no need to send 10 ul of template in individual tubes for each primer. Instead, please send one tube of template containing the following volume:
[# primers X (2 ul of template/primer)] X 1.5 = volume of template to submit |
Q. Can non-UAB investigators submit samples for sequencing? How much does a sequencing reaction cost for non-UAB investigators? A. Yes, we accept samples from non-UAB investigators. Cost is $10/reaction. |
Q. How often will I be billed for my sequencing? A. Invoices for sequencing are generated monthly and mailed to the PI entered in your user profile. |
Q. I have changed labs. Can I change my user profile? How do I do this? A. Yes, you can edit your user profile at anytime to update your records when you log in. |
Q. I am familiar with submitting samples for gel-based sequencing. Are there any differences in how I should prepare my samples for capillary-based sequencing? A. Yes. It is very important that the template be free of contaminants such as salt, RNA, and protein. Since our sequencer injects sample based on charge, any extra charged particles in the sample will reduce the overall signal of the reaction. It is also important to dilute templates and primers to the appropriate concentrations using water, not TE. If you are unsure of the concentrations of template/primer to submit, please refer to the table of recommended DNA concentrations for differently sized templates listed on our sequencing webpage.
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Q. When can I expect to get my results back? A. Samples received by 11AM are usually online the following morning. Sample submitted on Friday will be ready Monday morning. |
Q. I have cloned inserts into an siRNA vector. Since complementarity between siRNA and the targeted transcript is essential for RNAi, I need to sequence my construct. Do you have any tips for sequencing a hairpin? A. Yes, Ducat et al (Biotechniques, 34:10040-1144, June 2003) suggest finding a restriction enzyme which will cleave the hairpin, preferrably in the loop region, thus disrupting the inhibitory secondary structure. This will allow sequencing from the vector sequence across the stem of each side of the hairpin and off the cleavage site in the loop. Finding a restriction enzyme that cleaves with a 5' overhang will allow determination of the complete sequence. |
Q. Do you have any suggestions for success in sequencing GC-rich templates? A.There are a few tricks we can use in sequencing a GC-rich template but, the key is knowing in advance that the template is GC-rich. All the methods for sequencing GC-rich templates involve treating the sample differently than a standard sequencing reaction. One option is to run the sequencing reaction at a higher denaturation temperature. The second option is to add DMSO to the reaction in addition to using a higher denaturation temperature. Please let use know when you bring a template which is GC-rich (>65% GC) and then we can treat your sample appropriately to get you the best possible result.
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