Frequently Asked Sequencing Questions
Templates
Q: How should templates be prepared?
A: Plasmids should be prepared using a commercially available plasmid prep kit. Recommended: Promega Wizard Miniprep, Qiagen QuickLyse Miniprep, or Qiagen Plasmid Midi. PCR templates MUST be cleaned; Qiagen kits are recommended.
Q: How much template and primer do I need to submit for sequencing?
A: This amount depends on template type, the number of reactions required and the type of sequencing reaction.
- 1 Standard Sequencing rxn = 1ug of plasmid template or 100 ng for PCR product and 3.2ul of primer at 1uM.
Q: What are the best host strains?
A: Recommended: DH1, DH10B, DH5a, JM109, XL- Blue, and MV1190. Not recommended: JM101, HB101, TG1 & TG2.
Q: Should I combine my primer and template and submit them in the same tube for standard full service sequencing reactions?
A: No. Please submit all templates and primers separately in well-labeled microcentrifuge tubes. See submission guide for details.(link to NAC submission guide) (This does not apply to HT reactions.)
Q: Why can't I use TE or another buffer containing EDTA to submit my sample?
A: EDTA is a problem because it chelates the magnesium in a sequencing reaction.
Q: What concentrations should my template and primer be?
A: Concentrations should be 100ng/ul for plasmids, 10ng/ul for PCR products, and 1pmol/ul or 1uM for primers.
Q: How should I quantitate my DNA template?
A: Accurate concentration of your template is very important for a successful sequencing reaction. Using a spectrophotometer to read an OD alone is not recommended because it is not sensitive enough at typical plasmid concentrations, and contaminants such as genomic DNA, RNA, salts, and proteins all display some absorbance at 260nm. The best method is to use an OD260 and also run the sample on an agarose gel alongside standards of similar size and known concentration.
Q: How can I check my sample purity?
A: Both of the above methods (previous answer) should be used. A 260/280 ratio should be 1.7-1.9. Smaller ratios usually indicate contamination by protein or organic chemicals. An agarose gel will reveal the presence of DNA or RNA contamination. If salt is suspected, remove it using an ultrafiltration device, spin column or ETOH precipitation.
** Q: How can I get the worst possible data or no data at all?
A: Ignore all the above FAQs! BEWARE- POOR TEMPLATE quality = POOR DATA **
Data
Q: What is the difference between edited and raw data?
A: Edited data entails analysis of data by base editing through the evaluation of chromatograms, assembling of overlapping sequences to form consensus sequences, and primer design for extension. Raw data has not been analyzed.
Q: Why can't I see my primer in my sequencing data?
A: A sequencing read can begin anywhere from 30-100bp downstream from the primer-binding region.
Q:How can I open chromatogram files when I have NOT ordered online?
A: Applied Biosystems has freeware called Sequence Scanner Software v1.0 available to view, edit, and print chromatogram files. Visit http://marketing.appliedbiosystems.com/mk/get/sss_login to download the software. This link can also be emailed to you upon request.
Q: If I have specific questions/concerns about my data, what is the best time to address them?
A: Because the Core lab has a small staff and high workload, it�s best to call and set up an appointment time for detailed questions.
Sequencing Services
Q:What are HT reactions and how do I submit them?
A: HT or high-throughput reactions are reactions that are submitted in a Core approved 96-well plate. You must have at least 16 reactions to submit in this format. Submit reaction components (template+primer) following the required amounts in each well (listed in above). Please adjust your concentrations so that the volume being submitted is 7uL. Users are responsible for meeting required template/primer guidelines. If our control produces good data and the submitted samples fail, we assume no responsibility for failure.
Q: What are Ready to Run Reactions?
A: R2R of Ready to Run reactions are reactions where the user has prepared and run the sequencing reaction with ABI BigDye chemistry, completed a dye-terminator clean-up, and samples are submitted in HiDi. These reactions must be submitted in an ABI 3130 compatible 96-well plate. Please indicate what version of BigDye chemistry was used next to the Ready to Run check box on the Sequencing Project Form if not ordering online.
Policies
Q: How are failed reactions handled, and will I be charged?
A:Failed reactions may produce some readable sequence that can help indicate what the problem is. It is our policy to re-run any failed reactions that resulted from our error at no cost. If however, it is determined that template/primer quality or concentration was the problem, the PI will be billed for raw data.
Q: Should I cite the Core lab in a publication?
A: Yes, please cite the NAC in any publication that has data obtained from sequencing in our facility. Citations are used as an evaluation tool for Core facilities on campus and affect our funding, so please support us and help us to continue our services. Use the following as a guideline: Sequencing was performed at the Nucleic Acid Core Facility at UTHSCSA or Sequencing reactions were run on a ABI 3130xl at the Nucleic Acid Core Facility at UTHSCSA.