We do a lot of molecular cloning in the lab. Sarah has been working on a great protocols.io page dedicated to writing up the entire process, which I’ll link to once it’s completely set. But once you’ve successfully extracted DNA from individual transformants, a key tool in the molecular cloning pipeline is to find the clonal DNA prep that has the intended recombinant DNA in it. We use Sanger sequencing to identify those clones. That is what this instructional post will be about.

First, gather all of your data. I have a plasmid map folder on the lab google drive, where all of the physical .gb files for each unique construct is stored. Create a new folder (named after your .gb file), and *COPY* all of the relevant Sanger sequencing traces (these are .ab1) files into that folder. That will just help organize everything down the road. *PREFIX* the Sanger sequencing files with the clone id (usually “A”, “B”, or “C”, etc). This will make things easier to interpet down the road.

You presumably already have this plasmid map on Benchling, since that’s likely where you designed the plasmid, so open that up. if you don’t already have it on there, then import it. Once open, click on the to the “alignment” button on the right hand side.

If this is your first time trying to align things to this plasmid, then your only option will be to “Create new alignment”; press this button.

Next you’ll get to another screen which will allow you to add in your ab1 files. Click the choose files button, go into the new folder you had made in the “Plasmid” directory of the lab google drive, and import the selected abi files.

If you’ve successfully added the abi files, then the screen should look like this:

The default settings are fine for most things, so you can go ahead and hit the “create alignment” button. It will take a few seconds, but at the end you should get a new screen that looks like this.

The above screen is showing you your template at the top, as well as the Sanger seq peaks for each of your Sanger runs. If you’re trying to screen miniprep clones to see which prep might have the right construct, then go to the part of the map / alignment that was at the junction. For example, in this above construct, I had shuttled in the iRFP670 in place of mCherry in the parental construct, so anything that now has iRFP670 in there in place of mCherry is an intended construct. Looks like I went 3/3 this time.

This is a good chance to look for any discrepancies, which are signified by red tick marks in the bottom visualization. I’ve now moved the zoomed portion to that area, and as you can see, the top two clones seem to have an extra G in the sequence triggering these red lines. This is where a bit of experience / intuition is important. Since this is toward the end of the sequencing reaction and the peaks are getting really broad (compare to the crisp peaks in the prior image), the peak-calling program was having a hard time with this stretch of multiple Gs, thus calling it 4 Gs instead of 3. I’m not concerned about this at all, since it’s more of an artifact of the sequencing rather than a legit mutation in the construct. If we were to sequence this area again with a primer that was closer, I’m almost sure all of the constructs will show they only have 3 Gs.

Since all three constructs seem to have the right insertion any without any seemingly legit errors (yet), I typically choose one close and move forward with fully confirming it. All things being equal, I make my life simpler by choosing the clone that is earliest on the alphabet (so A in this case). That said, if all three looked fine but Clone C had by far the highest quality sequencing (say, the Clone A trace only went 400nt while the Clone C trace went 800+, I’d instead go with Clone C).

Next is sequencing the rest of the open reading frame. if you already chose a sequencing primer, then you’ve probably already attached a bunch of primers onto this map. But let’s pretend you haven’t already done this. To do this, go to the “primer” button and hit “attach new primers”, as below.

You may already have a primer folder selected. In that case, you’re all set to go. Otherwise, you’ll have to select the most recent primer folder. Click the “Add locations” area, open the triangle for the folder that says “O_Primers” and select the most recent folder (since this is currently august, this would be “20200811”. If selected, it should show up in the window, like the below picture.

Hit “Find binding sites” and it will come up with a long list of primers *that we already have* that are located in your construct.

This part gets kind confusing. To actually use these primers, you’ll first have to hit the top left box, located on the header row of the big table of primers. You should then see a check mark next to every primer.

Once you do that, hit the “Attach selected primers” button that it’s in green at the top right of the screen.

Once you do that, all of the primers that were previously listed but colored white before should now be colored green. NOW you’re free to switch back to your alignment.

Once you switch back to your alignment, the top should have a big yellow box that says “Out of Sync” and have a blue button that says “Realign”. Hit the realign button, which will call up one of your earlier screens, and you just have to hit realign once more.

Now all of the attached primers should show up in the top row of the alignment window. Since I have so many overlapping primers, this eats up a bunch of the space on the screen (you can turn off the primers if needed by clicking on the arrow next to “Template” and clicking off the box next to Primers). Still, now you can move around the map and find primers that will be able to sequence different parts o the plasmid you have not sequenced yet. In this case, I’ll likely just move forwards with one clone (such as Clone A), and I’ll start sequencing other parts of the ORF, such as the remaining parts of mCherry which I can likely get with the primer KAM1042.

Congrats, you’re now a Sanger sequencing analysis master.