UCR

Institute for Integrative Genome Biology



Sanger (BigDye) Sequencing Services


Genomics Services

DNA SEQUENCING SERVICES
Sanger (BigDye) Sequencing Services

Protocols
Preparation of Templates

Methods of purification of high-quality templates include QIAprep products (from QIAGEN) which include individual columns, strips of 8, or 96-well plates. These silica membrane products adsorb high salt contaminants producing DNA templates in water or low-salt buffer. There is also Promega Wizard and many other products including Fisher (Life Technologies) GenJet PCR Purification or  Zymo Researchto name a few.

For tips and trouble shooting of DNA templates, see Automated DNA Sequencing Chemistry Guide Section 3.

Submitting Templates for Sequencing
  1. All for forms DNA sequencing are on-line at the IIGB Billing and Reservation Site (http://iigbreservation.ucr.edu/). If you have not used the site previously you will need to register to access forms and other information such as pricing. Online forms must be filled out completely. A printed copy of your form should be submitted along with your samples to the Genomics Core. We are located at 2016 Keen Hall. For those who are off campus, please include a printed copy of the form with your shipped samples.
  2. If you are submitting samples in tubes, the tubes must have your full initials on the cap and a simple identifying number. For example, MBC1, MBC2, etc. Some other important tips:
      • We get many samples to process so for your own benefit, please put your identifying number on the tube, not just the cap which can break off easily.
      • Please be sure that your initials or other short identifier on the tube cap matches those found in the first column of the on-line submission form; this greatly reduces confusion when we set up the sequencing reactions.
  3. Small numbers of individual samples. For investigators submitting plasmid or PCR templates using their own primers: We recommend as a general guideline to add 3.2pmol of primer to your template in a total volume of 12ul water in a 0.2ml tube. Directions are incorporated into the on-line submission form, which should be filled out completely. This is twice the volume we need for a single reaction which allows us to quickly do a second reaction if the investigator and our staff decide that this is warranted. For recommended amounts of template, see Recommended Amounts of Templates and Primers for DNA Sequencing. We stock a selection of commonly used primers that we will add upon request at no additional cost. Available primers are listed below. Please submit templates in flat-top PCR tubes so that the sample name can be written on the top with a fine-tipped Sharpie. Place your tubes in one of the orange racks in the refrigerator indicated in the Genomics Core lab (2016 Keen Hall) and place your printed version of the submission form in the letter file indicated.  That’s it!

    For our safety and the safety of your results, please use tubes with caps can be removed relatively easily. Excessively tight caps can result in your samples being lost due to splatter when the lids are opened.
  4. Larger-scale sequencing projects in 96-well plates. This format reduces our costs by streamlining sequencing reaction setup and purification. The result is a lower price per template for customers.

    For this format, templates plus primers are submitted in 6 µl water. The quantity of template and primer and the volume of water are half of those submitted in tubes. Investigators must add primers if submitting in this format. For recommended amounts of template, see Recommended Amounts of Templates and Primers for DNA Sequencing. The amounts should be scaled accordingly. Plates should be filled by columns (ie, A1, B1, C1…BA2, B2 etc) and not by rows. Again, there is an on-line submission form. Please fill this in completely and provided a printed copy of the form when submitting plates for sequencing. Place your plates in the refrigerator indicated in the Genomics Core lab (2016 Keen Hall) and place your printed version of the submission form in the latter file indicated. That’s it!.

    Please use semi-skirted PCR plates with a single notch at upper right corner such as Thermo Scientific AB-1400 or similar plate. Seal the plates with an adhesive film to prevent evaporation and cross contamination.

NOTE: Plates must contain at least 48 samples to receive preferred pricing. We recommend that sample identifiers include investigator initials followed by an incremental number that will provide a unique identifier among plates submitted over time (ie, JQS1-1, JQS1-96, JQS2-1 etc). This reduces the chance of errors in our data collection software. As an alternative for those investigators needing identifiers associated with sequencing files, please inquire about sending an Excel file containing sample names by email to the Genomics Core; this requires a specific format which we will provide as a template by email. Briefly, the first column of the spreadsheet should contain well identifiers from the plate, top-to-bottom and left-to-right (A1, B1…H11, H12). The second column should contain your sample identifier. Send the spreadsheet as an attachment by email to gencore@ucr.edu on the day you submit your plate. We will import your file into our data collection software eliminating entry mistakes associated with complex identifiers.

Commonly Stocked Sequencing Primers

Primers are added free of charge for samples submitted in individual tube format.
 
T3                  5’-AAT TAA CCC TCA CTA AAG GG-3’
T7                  5’-TAA TAC GAC TCA CTA TAG GG-3’
M13F(-21)    5’-TGT AAA ACG ACG GCC AGT-3’ (used for M13F without note)         
M13R            5’-GAA ACA GCT ATG ACC ATG AT-3’ (used for M13R without note/pGEM)               
SP6               5’-ATT TAG GTG ACA CTA TAG-3’

Other available primers:
pTV                5’-TTT TTT TTT TTT TTT TTT TTV-3’ (to overcome poly A in template)
                       5’-AAA AAA AAA AAA AAA AAA B-3’ (to overcome poly T in template)
pCD               5’-CCC CCC CCC CD-3’ (to overcome poly G in template)
pGH               5’-GGG GGG GGG GH-3’ (to overcome poly C in template)
M13F(-40)     5’-GTT TTC CCA GTC ACG AC-3’
M13R(-24)    5’-AGG AAA CAG CTA TGA CCA TG-3’ (For pENTR/pDONOR)     
pET-UP         5’-ATG CGT CCG GCG TAG A-3’{For pET from Invitrogen (167~181)
                                                                                                not from Novagen}             
T7-TERM      5’-GCT AGT TAT TGC TCA GCG G-3’ (For pET)

NOTE: "V”=A, C or G; "B"=T, C or G; "D"=A, T or G; "H"=A, T or C.

Data Retrieval

Following sequencing, data files are transferred to the Genomics Core Dropbox account. Customers are emailed a link to the data for secure download using any common web browser such as Internet Explorer, Safari or Firefox. Data download does not require customers to have their own Dropbox accounts.

Viewing Sequencing Results

Your results will be **.abi files, and there are several very good free sequencing viewers.

  • For PC users, you can download Chromas or FinchTV. An especially good viewer is Sequence Scanner v1.0 from Life Technologies. Only v 2 as a demo is available free from Life. However, v 1.0 can still be found on the web for download. In addition to the traces, you can also obtain run parameters generated by the ABI sequencing to help trouble shoot your sequence runs.
  • For Mac users, you can download FinchTV.
Sequencing from Plasmid Preps in 96-Well Format

The Genomics Core offers this service at a modest cost. The service entails users providing a 96-well block containing cells grown in 1ml of medium under the appropriate selection. These are incubated overnight in our facility. The following day, our staff will prepare 96-well format plasmid prep which is then subsequently sequenced using one of our in-house primers as specified by the user. For plasmid preps we use the Perfectprep Plasmid 96 Vac Kit (5 PRIME) which utilizes alkaline lysis following by DNA binding and washing. For more information, see the (5 PRIME) website. Please download and print our Request for Plasmid Preparation and DNA Sequencing Form to submit samples as needed.

NOTES:

  • UCR and external customers MUST contact Core Staff at least 24 hours prior to their desired drop-off time. We have a limited per day capacity, and those who bring plates without prior arrangement risk having their sample preps delayed.
  • The Genomics Core will supply sterile 96-well blocks to customers for plasmid preps. These blocks are sterile and fit our filtration equipment.
  • Under ideal conditions, sequence data will be posted 3 to 4 business days from the date of dropoff. However, depending upon our workload, your results may be posted later. So please do not assume your results will be posted in 3 business days. Can you also always contact us, and we can provide an approximate posting date.  Thanks for your understanding.  
Processing Time

We set up sequencing reactions five times a week. Samples received after the cutoff time on Friday are processed on Monday. We will provide data within two working days. The data will be posted to our Dropbox account as soon as possible, often the next working day after templates are received. Emails with Dropbox links will inform clients that data is ready for download. These processing times are applicable to samples received prior to 3:45PM. Samples received after that time may not be processed until the following business day.

We strive to provide a two-day processing time or faster. However depending upon our workload and whether we receive a minimum number of samples per plate by our cut off time each working day, your results may take longer to process.

 Technical Tips (Troubleshooting)

We are asked frequently by users why their DNA sequence is not of good quality. This is not always easy to diagnose based solely on a chromatogram. However, common problems are primers that are not specific, templates of insufficient purity or template amounts that are not quantified. It is also common for templates to be mixed resulting in unreadable chromatograms. The staff at the Genomics Core is always happy to discuss any sequencing problems and if possible to provide suggestions. We will do our best to assist you, but please remember that we sequence the materials you provide to us. It can be difficult to diagnose problems due to cloning or amplification problems. We encourage customers to read the following materials which provide many useful suggestions.

Automated DNA Sequencing Chemistry Guide Chapter 8 (Troubleshooting) from Applied Biosystems.

  • This is a very useful guide and will help users to understand sources of potential problems based on interpretation of chromatograms and potential solutions. 
  • SEQanswers  is a website that covers mostly nextgen sequencing such as Illumina. However there are also many questions and answers pertaining to Sanger sequencing and sample prep of PCR and other templates. You can also post a question there for the community.

More Information

General Campus Information

University of California, Riverside
900 University Ave.
Riverside, CA 92521
Tel: (951) 827-1012

Career OpportunitiesUCR Libraries
Campus StatusDirections to UCR

Genomics Information

Institute of Integrative Genome Biology
2150 Batchelor Hall

Tel: (951) 827-7177
E-mail: Aurelia Espinoza, Managing Director

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