Sanger Sequencing Guide

This tells the SOP for Sanger Sequencing at UC Davis.

Generally, sequence 2-4 clones of every vector you intend to use & save down the E. coli in glycerol stocks.

Ideally sequence only after you have done colony PCR to confirm the vectors have an appropriately sized insert.

Submitting sanger sequencing runs

There are two options. We prefer Eurofins due to the quality and cost. However, we need to have at least 9 samples in order to get the free shipping.

If you just have a few reactions, you should use the UCD college of Biological Sciences DNA Sequencing facility: http://dnaseq.ucdavis.edu.

Prepare your sample (Eurofins)

This is general steps because Eurofins could update their process. So look at our Eurofin’s Sanger instructions. https://eurofinsgenomics.com/en/products/dna-sequencing/seq-sample-submission-guidelines/

Our Eurofins login information is in the “Ordering” google sheet within our lab shared Google Drive

• Log your samples into the Eurofins submission site.
• Apply the Eurofins barcodes to each tube. (These are free. If we run low, order more from Eurofins)

• Add appropriate volume of plasmid to the tube. If you are using the common sequencing primers, you can have Eurofins add their own. If using specific primers, follow Eurofin’s instructions.

• Put all tubes inside a zip lock bag. Put inside a small box with the Eurofins paperwork. Secure tubes with packing material or tape the ziplock into the box so it doesn’t rattle around (tubes could open). Note: when possible, reuse ziploc bags / small boxes that we receive materials in.

• Bring package to Hutchison Mailroom (Rm 361) before 8:30 AM to ensure UPS picks up.
  • Outgoing packages are placed inside the room on floor in the area immediately to the right of the door - big sign on wall says “FedEx and UPS Pick Up”Bring the box to the UPS pick-up location in the Mail room on the 3rd floor. You can put the package in the mailroom overnight. Plasmid DNA should be stable at room temperature for a while.

• Eurofins will email you a link to your data within 1-2 days.

• After you get the sequences, check the quality. We are allowed to request sample re-runs for 10% of an order for free. If you have < 500 bp of usable sequence, request a re-run on up to 10% of your samples. Forward your order information to GenomicsSupport@eurofins.com

Sequencing with the Sanger Sequencing service on campus

(This might be incomplete)

• Follow guidelines on Website: http://dnaseq.ucdavis.edu/SampleSubmission.cfm. Brief things:

1. Primers (4 ul / sequencing run; 3 uM concentration*) and template DNA (6 ul / seq run; concentration depends on size ) must be in 0.5 ml tubes with clear labels.
   • *Dilute the 10 uM working stocks of primers. e.g. 9 ul of 10 uM stock + 21 ul H2O = 30 ul.

• Submit an order: http://dnaseq.ucdavis.edu/orders/. You’ll need an account, a

• Place in a ziplock bag & label with your name & the order number (generated when you submit)

• Drop off reaction in 0280 Storer Hall (basement in the building connected to Hutchison)

Submitting sanger sequencing runs externally

Sequence with Eurofins Genomics: https://eurofinsgenomics.com/en/home/

Prepare your sample:

Drop off your sample:

What to do after you’ve downloaded the sanger files

probably .ab1 traces

Save them into a logical folder (e.g. a “Sanger Results” folder in your Cloning folder)

Benchling Approach

This approach allows you to overlay the sequence and raw trace with your DNA-of-interest

• Open your plasmid plan in Benchling (you made one right ಠ_ಠ)
• Click the Alignments tool on the right > Create New Alignment > Choose Files (Choose all ab1 files that map). > Create Alignment
• Inspect the Alignment for sanger seq quality
  • Does it match at all? Bottom of the screen will be a diagram showing grey=match and red =mismatch.
  • How was the sequencing quality (abunance of “Ns”, which will print as red at the bottom & also raw traces–are there clear & equally spaced peaks)?
    • If sequence is gibberish (entirerly or <200 bp usable sequence) email the seq center and request a free rerun.
• If sequencing quality is good… look for problems.
  • Gibson assembly can create Indels at junctions of fragments. Inspect those carefully if it’s important.
  • Look for SNPs (small red areas in grey area on bottom bar).
    • Inspect raw trace. If it is an N, make your own judgement call. Rename bases with lowercase bases if you think the .ab1 basecall was too stringent.
• Decide whether you have the “correct clone” (make an overnight & save it in -80) or whether you need to sequence additional clones.