DNA sequencing

Key Concepts

Now we want to check if the plasmid contained in our bacterial strain is correct. Common problems that we want to check for: 1) The bacteria were transformed with an empty vector that did not contain our GFP insert, 2) the insert contains a non-synonymous nucleotide substitution due to PCR error. 

We will be taking our sequencing reactions to a sequencing facility on campus. Typically, the sequencing will happen overnight and our sequencing files will be emailed to us the next day. Remember, for sequencing you only add 1 primer (in contrast to PCR in which you add 2).

Materials

• Purified plasmid from miniprep
• Sequencing primer
• Nuclease-free water
• 1.5mL microcentrifuge tube

Procedure

Mix your purified plasmid and primer in a 1.5ml microcentrifuge tube according to the sequencing center’s specifications: 

Plasmid sample submission = 0.5 µg DNA/5kb DS plasmid + 8 pmol primer in 13 µL

1. Add the appropriate volume of purified plasmid based on the equation above and the concentration of your miniprepped plasmid to a 1.5mL microcentrifuge tube. Do not add more than 10uL of plasmid.
2. Add 3 µL of 1 µM T7F sequencing primer.
3. If the final volume in the tube is less than 13uL, add nuclease-free water to bring the final volume up to 13uL.

Once you have mixed your plasmid (miniprep), primer and water in a tube, label the top of the tube with your number assigned to you by the instructors. We’ll be submitting the sequences with a form that will have the reactions labeled this way, so please follow this format exactly.

Sequence analysis:

We will use Benchling to analyze our sequencing data. To do this, make an account on benchling.com and request to join the ‘QB3 Bootcamp’ group (or another group, as instructed). This will give you access to the sequences to compare.  You can use Benchling to catalog sequences, primers, and cloning projects in your own labs, not just in this Bootcamp! Benchling has several tutorials to introduce you to the variety of features that we will not be covering in this class.

From your Benchling homepage go to the QB3 Bootcamp page (right side of homepage) and open the GFP cloning folder. This folder contains two sequences: 1) the parent vector pET28, and 2) our desired construct, pET28 His6-GFP.  We will be making an alignment of your sequencing files to the pET28 His6-GFP file. 

Once you have opened the pET28 His-GFP sequence you’ll see two representations of the vector: on the left a sequence level view of the entire plasmid and on the right, a schematic. This plasmid has already been annotated with features such as where the open reading frame for GFP is located.

Create an alignment of your GFP cloning .ab1 file by clicking on the Alignment icon (appears as four little bars on the right-hand toolbar). This will prompt you to create an alignment. Choose your “1_your number_G.ab1” file. This file contains both the sequence data and the raw chromatogram from the sequencing run. The other sequence contains only the sequence without the chromatogram.  The assignment of peaks is automatic but not always correct, so if you see a mutation you’ll need to double check the assignment of the base against the chromatogram trace.

Was GFP successfully inserted in your sample? Were there any mutations?