protocols

Lab protocols for the Lowe-Power lab (scroll down for table of contents Readme)

View the Project on GitHub lowepowerlab/protocols

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

Sequencing with the Sanger Sequencing service on campus

(This might be incomplete)

  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.

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