NEB Podcast #54 -
Transcript
Interviewers: Lydia Morrison, Marketing Communications Writer & Podcast Host, New England Biolabs, Inc.
Interviewee: Rachel Carver-Brown, Senior Technical Support Scientist, New England Biolabs, Inc.
Lydia Morrison:
Welcome to Lessons from Lab and Life Podcast, brought to you by New England Biolabs. I'm your host, Lydia Morrison, and I hope this podcast offers you some new perspective.
Today, I'm joined by NEB Senior Technical Support Scientist, Rachel Carver-Brown. Rachel's here to join me, to kick off our molecular cloning series, and today, we'll be talking about mutagenesis, both site directed mutagenesis and multipoint mutagenesis, and the products that NEB offers to help support this.
Rachel, thanks so much for coming down to the studio today.
Rachel Carver-Brown:
Thanks so much for having me. I'm excited to be here.
Lydia Morrison:
So Rachel, tell our listeners what mutagenesis is, and why a researcher might want to use it.
Rachel Carver-Brown:
Mutagenesis is making a specific change of a plasmid sequence. It is adding in some perhaps a His-tag into the plasmid, or making a change in the sequence to change which amino acid is at a given position. Researchers would want to use this for those reasons, but also, it can be used to change the sequence and add a restriction site into a plasmid, or actually even change it to remove a restriction site. Which that's helpful, I know, in the NEBridge workflow, those are reasons why mutagenesis is often employed. It's mostly just making a change to the sequence that's there.
Lydia Morrison:
And are there different ways of accomplishing mutagenesis?
Rachel Carver-Brown:
There's two main methods to accomplish a site directed mutagenesis, and the differences there lie in the types of primers that are used. One method that's used, the common QuikChange® method, uses overlapping primers. And those overlapping primers result in the double-stranded plasmid at the end that with nicks, and that is then taken into transformation, and the cells seal those nicks.
The other method, and the method that our Q5 Site-Directed Mutagenesis Kit employs, uses back-to-back primers. These primers, they have five prime ends, butt up against each other, and that's where mutations are introduced. And they'll amplify in opposite directions around the plasmid. That results in an exponential amplification, which is much more efficient than a linear amplification you would get with the overlapping primers.
Lydia Morrison:
Could you explain a little bit more about why our kit uses the back-to-back primers that you mentioned, instead of overlapping primers? And a little bit more about sort of how those work?
Rachel Carver-Brown:
Sure. The back-to-back primers, the main reason that our Q5 Site-Directed Mutagenesis Kit uses the back-to-back primers is because it allows for a much more efficient and exponential amplification.
So their primers, essentially, if you think about it, they're sitting at like 12 o'clock on a clock face, and they'll go down around in opposite directions. And that ends up with a linear product, which can then be exponentially amplified with those primers as the PCR cycles continue.
The overlapping primers, because of how they are oriented and how far around the plasmid they'll go, they actually only have a linear amplification. That matters, because the linear amplification, you need to start with a lot more material than you would with an exponential amplification. So having a large amount of plasmid in there to start can actually be difficult for PCR reactions having too much input. Whereas, with the exponential, starting with a very small amount of plasmid, that PCR reaction runs more efficiently.
Lydia Morrison:
Understood. So it sounds like it kind of solves for a couple of problems. Speaking of problems, what are the most common problems that customers face when they're using the kit?
Rachel Carver-Brown:
The most common problem that I hear with the Site-Directed Mutagenesis Kit is ending up with wild type colonies at the end. You go through all the effort to make the change, and the result actually isn't changed. That's the most common issue that I'll hear about, and it's actually something that is fairly easy to investigate and figure out where the problem lies.
Lydia Morrison:
And how do you go about that?
Rachel Carver-Brown:
The first thing to do would be to check and make sure that what you're amplifying in the PCR step actually is what you're intending to. So if you run the PCR product on a gel, and there's no amplification at all, you don't get a band, then that's your problem. And then we just work together and figure out the PCR reaction and get that working.
Lydia Morrison:
So is it always necessary to run the PCR amplified fragment on a gel prior to using the DNA in a workflow?
Rachel Carver-Brown:
It's not necessary to run it every single time. If you're working with a similar set of primers that you've used before, or if you are doing a high throughput, you may not want to screen every single primer set. But certainly, if you run into wild type colonies or issues downstream, that is probably the first place to check, and the first troubleshooting step I'd recommend.
Lydia Morrison:
Can researchers use homebrew competent cells in this workflow?
Rachel Carver-Brown:
They can, yes. We do have two versions of our Site-Directed Mutagenesis Kit. One includes our NEB 5-alpha high efficiency comp cells, but we do have a version without comp cells. So researchers can definitely use their own if they were high efficiency, and cells that work well in their workflows in the past, they can definitely use those.
Lydia Morrison:
And how accurate is the Q5 Site-Directed Mutagenesis method for generating desired mutations, or indels?
Rachel Carver-Brown:
The accuracy of the kit is kind of twofold. It uses the Q5 high-fidelity polymerase, so the high-fidelity aspect of that, its proofreading activity and its innate accuracy in copying the plasmid sequence is great, and gives very high accuracy in that regard. In terms of the number of colonies at the end of the reaction, assuming PCR worked well, typically we'd see at least 75, if not 80% of correct mutagenized colonies.
Lydia Morrison:
Wow. That's pretty high efficiency, pretty high odds.
Rachel Carver-Brown:
It is pretty high. Our QC standards are pretty high for the kit, and our customer experiences that I've heard, it's matching for them. Customers really like using the kit.
Lydia Morrison:
Okay. So what about multi-site mutagenesis, as opposed to a single site mutagenesis? Would someone do multiple rounds of site directed mutagenesis, or is there another way to accomplish that?
Rachel Carver-Brown:
If a researcher is wanting to make multiple changes within the same plasmid, they certainly could use the Q5 Site-Directed Mutagenesis Kit, and make those changes sequentially. So you go introduce the first change, go through the whole process, the KLD, the growth, the transformation, and then use that resulting plasmid in the next round, and the next round, and so on. That does take a lot of time, though. And another way to go about it would be to use NEBuilder HiFi Assembly Kit. The HiFi Assembly allows you to put together multiple fragments, and you can actually introduce those mutations in the overlaps of that workflow. So those multiple mutations would be introduced on each fragment, and would be able to be assembled all in one reaction, in one go.
Lydia Morrison:
Well, that seems a lot more efficient.
Rachel Carver-Brown:
It is, yes. And it saves a lot of time, and a lot of people are moving to that for multi-site mutagenesis.
Lydia Morrison:
Yeah. Does it end up saving materials too? Like overall it saves time. I mean, I guess it would end up saving like comp cells in terms of growth and time for that.
Rachel Carver-Brown:
It would definitely save comp cells as well. Yes, because not having to do the growth each time as you would with the sequential SDM Kit reactions. Saves on amplification reagents too in some ways, because you're not necessarily having to go through and troubleshoot that as often as well.
Lydia Morrison:
Gotcha. So Rachel, before I let you go, what are your top tips for a researcher who's trying to perform mutagenesis?
Rachel Carver-Brown:
It's a great question. One of the first things I'd suggest, is to use our NEBaseChanger primer design tool, that helps make the primer design process of those back-to-back primers much more streamlined. And we actually have a new version of the NEBaseChanger tool that enhances capability. You can use it in batch mode, and it's a lot clearer in terms of making amino acid changes. That new tool, we'll have a video tutorial coming up soon, hopefully, to help you walk through it even more. But if you do have questions, you can always reach out to tech support for that as well.
Lydia Morrison:
Awesome. Anything else?
Rachel Carver-Brown:
If it's the first time with a kit, the other thing I would suggest is running that PCR product on a gel, just to be comfortable and be confident in your first round through it. Again, it's not necessary, but for the first time, I would definitely suggest that.
The last tip I'd have is, if you do run into any problems with the kit, in terms of, you have wild type colonies at the end, which is the most common thing that they might run into, give it another go. But if after the two iterations it's still not giving you the results you need, call tech support. We're more than happy to help. We have a lot of scientists very knowledgeable on the kit that can help walk you through and troubleshoot with you, to get you the correct product at the end.
Lydia Morrison:
Awesome. Thank you so much for joining us today, Rachel. I think this will be a really informational podcast for people who are new to cloning and new to mutagenesis. And it's also a great reminder that we have amazing scientists providing tech support to our customers. So don't be afraid to reach out if you run into problems, because someone like Rachel will help walk you through all the solutions to make sure that you experiment is working successfully.
Rachel Carver-Brown:
Thank you so much for having me.
Lydia Morrison:
Thanks for joining us for this episode of the Lessons from Lab and Life podcast. I'd like to take a moment to remind you where to find the helpful resources we mentioned in this conversation. Head over to our website NEB.com, to find videos and animations, as well as the answers to some questions our tech support team frequently receives.
If you need to reach tech support yourself, you can email info@NEB.com within the US. And if you're outside of the states, please contact your local NEB subsidiary or distributor to receive help from our knowledgeable global technical support team.
Please be sure to tune in next time for our second episode of the Molecular Cloning series, when we'll be talking about Golden Gate Assembly. Golden Gate Assembly is a seamless DNA assembly technique that can be used to join many, many fragments of DNA in a single reaction.
