Genomics Services

DNA SEQUENCING SERVICES
Sanger (BigDye) Sequencing Services

Protocols
I. Preparation of High Quality Templates

Methods for 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 Research to among others. Note that the following contaminants can negatively impact sequencing.

Contaminant             Max level of tolerance          Effect

RNA                                  1 ug per reaction                         high background

PEG                                   0.3%                                              polymerase inhibition/run failure

EtOH                                 1.2%                                              polymerase inhibition/run failure

NaOAc                               0.4mM                                          polymerase inhibition/run failure

Phenol                                0                                                    polymerase inhibition/run failure

Chloroform                        0                                                    polymerase inhibition/run failure

CsCl                                    0.4mM                                          polymerase inhibition/run failure

EDTA/salts                       0.2mM                                          injection failure/weak signal

II. Submitting Templates in Tubes for Sequencing

1. Forms. DNA sequencing forms are on-line at the IIGB Billing and Reservation Site. 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. Tube Samples (Plasmid or PCR templates using their own primers) ($4.75 per sample). We recommend adding 5 – 10pmol 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 a second reaction if this is warranted. For recommended amounts of template and primer, see Recommended template amounts.

3. Tube samples (we add primers) ($4.75 per sample). We stock a selection of common primers that we add upon request at no additional cost to samples submitted in tube format. Available primers are listed below. Primers are added free of charge for samples submitted in individual tube format. Recommended templates without primers should be submitted in a total volume of 10ul (rather than 12ul) according to Recommended template amounts.

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 primers:
pTV                5’-TTT TTT TTT TTT TTT TTT TTV-3’ (to overcome poly A in template)
pAB                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.

4. Submitting your samples. Submit samples in flat-top PCR tubes with the sample name written on the top with a fine-tipped Sharpie. We process lots of samples. Tubes must have full initials on the cap and a simple identifying number. For example, MBC1, MBC2, etc. Also write your identifying number on the tube, not just the cap which can detach easily. 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 during sequencing reaction setup. Place your tubes in one of the orange racks in the posted refrigerator in the Genomics Core lab (2016 Keen Hall) and place your printed version of the submission form in the letter file next to the refrigerator. 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.

III. Submitting Larger-scale sequencing projects

1. Plate format (48 or more samples at one time) ($4.00 per sample). Although we will accept 48 or more tubes at this same lower price, providing templates in plates reduces costs by streamlining sequencing reaction setup and purification. The result is a lower price per template for customers. For this format, recommended amounts of template and primers are submitted in 6 µl water or Tris per well. The quantity of template and primer and the volume of water are half of those submitted in tubes (see Recommended template amounts). Investigators must add primers if submitting in this format. Please use semi-skirted PCR plates with a single notch at upper right corner such as VWR 83007-374, Thermo Scientific AB-1400 or similar PCR plate. Seal the plates with an adhesive film to prevent evaporation and cross contamination. 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 for Plate Sequencing. Place your plates in the orange racks in the Genomics Core lab (2016 Keen Hall) and place your printed version of the submission form in the letter file. That’s it!

NOTE: Plates must contain at least 48 samples to receive $4.00 per sample 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, we will accept 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.

IV. Data Retrieval

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.

V. 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.  An especially good viewer is Sequence Scanner v1.0 from Thermo (Life Technologies). It cannot be found even on the Life website. 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 sequencer to help trouble shoot your sequence runs.
  • For Mac users, there may be some options such as MacSequenceView which costs $14.  Free options such as FinchTV have been discontinued. Alternatively, using Parallels for Mac will permit Windows to run in a separate partition.
VI. Other Options

1. Sequencing from Plasmid Preps in 96-Well Format. 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 sequenced using one of our in-house primers. For plasmid preps, we use a plate based vacuum kit which utilizes alkaline lysis following by DNA binding and washing. 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.
  • The Genomics Core will supply sterile 96-well blocks to customers. 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 drop off. 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.
VII. Processing Time

Typical turn around time is usually the next business day. Depending on the workload it may be two days, but we do our best for one-day turnaround. Cut off time each day is 3:45PM. Samples received after the cutoff time will be processed the next business day. Samples received on Friday are processed and posted on Monday. The data will be posted to our Dropbox account as soon as possible after templates are received. Emails with Dropbox links will inform clients that data is ready for download.

We strive to provide one-day processing time. However depending upon our workload and whether we receive a minimum number of samples per plate by our cut off time each working day.

 VIII. 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:

1. Primers that are not specific leading to mixed sequencing traces

2. Primers that are not pure an result in N and N+1 overlapping sequences due to primer at multiple locations

3. Templates of insufficient purity and containing contaminants (see, I. Preparation of High Quality Templates)

4. Template amounts that are not quantified

5. Templates especially from PCR that are a mixture of products resulting in overlapping and unreadable chromatograms

The staff at the Genomics Core is always happy to discuss any sequencing problems and provide suggestions. The guidelines we provide on this web page are general. We will work with you to optimize your sequencing to provide the best quality possible. Please remember however 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.
  • Chapter 3 discusses DNA template preparation.
  • 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.