NEB Podcast #69 -
Transcript
Interviewers: Lydia Morrison, Marketing Communications Manager & Podcast Host, New England Biolabs, Inc.
Interviewee: Jack Martz, Technical Support Scientist, New England Biolabs, Inc. & Heejin Kim, Ph.D., Development Scientist, New England Biolabs, Inc.
Lydia Morrison:
Welcome to the Lessons from Lab and Life Podcast, brought to you by New England Biolabs. I'm your host, Lydia Morrison, and I hope this episode brings you some new perspective. Today I'm joined by an NEB technical support scientist, Jack Martz and NEB development scientist, Heejin Kim, to talk about best practices for DNA cleanup. We'll talk about the history of DNA cleanup and the methods available, as well as share some guidance on selecting the method that's right for your work.
Jack and Heejin, thanks so much for joining me in the studio today.
Jack Martz:
Thanks for having us.
Heejin Kim:
Thank you.
Lydia Morrison:
Yeah, I'm super excited to get into our topic, but before we dive into DNA purification and cleanup, could you introduce yourselves to our audience and just tell them a little bit about what it is you do here at NEB?
Heejin Kim:
Sure. My name is Heejin Kim, and I am a Development Scientist at New England Biolabs. I work on Monarch product lines for DNA extractions and purifications.
Jack Martz:
And I'm Jack Martz. I'm one of the Technical Support Scientists who supports our Monarch lines.
Lydia Morrison:
Amazing. I can't think of two better people to be in the studio today and to explain to our listeners the concept of nucleic acid purification and sort of what sets different methods apart. So let's start by defining PCR and DNA cleanup. Could you tell our listeners what that is and how you purify the DNA?
Heejin Kim:
Sure. DNA cleanup just refers to a process of purifying DNA to remove any contaminants or any unwanted components such as proteins, detergents, nucleotides, primers, and things like that. Commonly DNA cleanup is used after enzymatic reactions such as PCR, restriction digest, or even just to clean up DNA from any extraction that you have used to remove any impurities, so the purified DNA can be used for various downstream applications.
For the methods to purify DNA, actually, there are a few different options to achieve this. Common purification methods include one, phenol chloroform extraction. One of the most common ways to do this, or the oldest method, you can do this, and this method uses a mix of phenol and chloroform to separate the DNA from proteins and other contaminants. After spinning the mixture, the DNA stays in the aqueous layer, which can be collected for the further use.
Second method is ethanol precipitation. This method is simpler, but it's only uses ethanol to precipitate the DNA out of the solution using ethanol or isopropanol.
The third option is column-based purification. One of the most common ways to purify DNA. In this method, DNA just binds to a silica column with a chaotropic salt buffer and is washed to remove impurities.
The fourth option is the magnetic bead-based purification where DNA binds to a magnetic bead with various binding chemistries, which are then separated from the solution using a magnet and washed again to purify the DNA.
Lydia Morrison:
So I'm curious how DNA cleanup methods have changed over time. Is it sort of like a natural progression from phenol chloroform to ethanol precipitation to sort of the ubiquitous column-based methods that we see in the boxes on everybody's benches to magnetic beads? Or do some of these things kind of exist at the same time?
Jack Martz:
Both. You kind of had it right where things started with phenol chloroform extractions in the 1950s. Phenol chloroform extractions lead to high-quality DNA, but they're very hazardous to use. The chemicals, phenol and chloroform are toxic and you have to do it under a hood, which raised some concerns as to the risk of these type of extractions. They also worry about phenol carryover, which could inhibit downstream applications. So then ethanol precipitation kind of got popular in the 1970s was when it was developed. It's simpler than phenol chloroform extraction, but it is time-consuming and it's less efficient. It became a standard method due to its simplicity and how effective it was. But the typical prep can take anywhere from 30 minutes to an hour if not longer, and the results are variable in terms of your yield and purity. So in the eighties and nineties, silica columns really became popular when they were introduced.
So using a spin column format and a centrifuge, it's a lot faster. It's a lot safer to both phenol chloroform and ethanol precipitation and is used probably most commonly today and has been widely adapted in life sciences to purify DNA using really common lab equipment. It wasn't until the 2000s that DNA cleanup with magnetic beads was really developed. It uses a magnetic bead coated with functional groups that allow binding to DNA. They're very popular now because they're really, really high throughput and you can automate them. That all said, most people use all of the different methods to purify DNA. You will come across all of them still in a lab today.
Lydia Morrison:
So you said that column-based cleanup was one of the more popular varieties. That's certainly what I've observed. Are there advantages to using the column-based method over some of the other cleanup methods that you've described?
Heejin Kim:
Sure. As I mentioned, there are a few different ways to purify DNA, but column-based methods offer several advantages over other cleanup methods. For example, number one, speed and efficiency. It's just the process faster because it involves fewer steps compared to other methods, specifically compared to ethanol precipitation and phenol chloroform, methods, which can take anywhere from 30 minutes to few hours. A spin column allows for efficient prep taking maybe less than 20 minutes in general.
Second is, as Jack pointed out, the safety aspect of it. The spin column methods avoids using hazardous chemicals like phenol and chloroform. This really offers a safer option to purify DNA by avoiding use of this hazardous chemicals.
And the third advantage that I would like to point out is the purity of the DNA that you get. The spin column method provide high purity DNA, which is suitable for sensitive downstream applications such as sequencing or any other self-sensitive enzyme reactions. But this purity is, I would really like to point that it's really reliable method for the column-based methods because the purity can be an issue with other methods such as ethanol precipitation.
And this leads me to my fourth advantage that I would like to point out, which is the reproducibility. If you're a scientist on the bench, you can expect more consistent results by using column-based methods in both yield and purity across different samples, allowing spin column methods to be a very convenient and very reliable method to purify your DNA.
Lydia Morrison:
So I know there are lots of companies in the nucleic acid purification space. Do they all make their own columns and reagents?
Heejin Kim:
Actually, no. There are many different options and columns and kits with different features and advantages suited for different purposes for researchers. I can start by asking questions about the quality of the columns in the kit. Do you know if and how the columns was made to fit the extraction? Take NEB for example, since we utilize our state-of-the-art manufacturing automation process, we are actually able to manufacture our own uniquely engineered columns at high quality that is consistent. And then we offer these kits as well as providing them separately for the flexibility. So if the customers wants to just buy more columns, not the buffers, they just have the option for that. By manufacturing our own columns and buffers, actually, this really allowed NEB to build different technologies around this products and to meet different extraction needs and gain more deeper understanding of the products we're making. But I think it really doesn't stop there because this capability and expertise that we are building around these kits really extends our ability to really help the other scientists. NEB can drive science forward by providing recommendations, solutions, helpful tips and tricks, and even offering advice through technical support, modifying the protocols specific to the customer needs. And I think in the end, it's really all about contributing to the scientific community and making a real impact.
Lydia Morrison:
I love that. I think that all sounds amazing, and I really appreciate that NEB has put the time and thought into the engineering of these columns and how we can make them better. That being said, they are still plastic columns, right? So obviously we know plastic is bad for the environment, and we know that lab waste plastics are an issue in terms of laboratory research. Are there efforts to make DNA purification more environmentally friendly?
Jack Martz:
Oh, yeah, absolutely. As you referenced, the life sciences field has been really keeping an eye on plastic waste, especially in the recent years and across the board, there are ongoing efforts to reduce the environmental impact of all of the plastic that we use. We try to use less so reduce plastic usage, and we are also trying to recycle more. For DNA cleanup kits there are ways to just inherently reduce the plastic usage. For the Monarch kits, our bottles and our columns use less plastics and other plastic parts while still having the strength needed to survive spinning in the centrifuge. The packaging is also sourced for recyclability and using eco-friendly practices wherever we can. Other efforts in science to reduce environmental impact include active research on biodegradable materials, green chemistry, and then recycling programs that may be offered by companies and institutions.
It's important for scientific communities and companies to actively put an effort to reduce the plastic waste wherever we can, and also to put efforts on educating and providing resources to raise the awareness of reducing plastic waste and adopting sustainable practices as much as possible.
Lydia Morrison:
Yeah, definitely an important reminder, I think, and hopefully something that lots of bench scientists are keeping in mind as they're going about their daily work. I'm curious if you have any tips for helping customers choose the right purification methods for their project, for their work, for their research?
Jack Martz:
Yeah, absolutely. And we do this all the time on technical support. So there are a few things to keep in mind. The first is to define what exactly you need for your research. This means really understanding what your samples are, what you're going to purify, and if that nucleic acid is there. So are you looking for double-stranded or single-stranded DNA? Is it circular? Is it linear? Are you looking for small fragments of DNA or very, very large ones? You then want to check if that is compatible with what you're using and if our kits are compatible with your downstream applications. So keep in mind the yield that you need, the capacity of the column that you're using. Does the kit that you want to use efficiently remove contaminants that you are trying to get rid of and recover enough DNA for your downstream application?
Some other things to keep in mind are do you need high throughput or do you need automation? If you need to clean up large amounts of DNA samples on a regular basis, you might consider high throughput or automation compatible kits like our MAG kits. Automated systems can save time and reduce errors.
You can also use your resources, so if you're having trouble with either finding a right kit for your sample or you need support to have successful preps, you can use the resources available that we have. Typically, product webpages, FAQs manuals, provide information that might help you get a successful prep. But if you have specific questions, you can always reach out to technical support, and we're happy to guide you to make sure that you have success with our kits.
Lydia Morrison:
Well, I want to thank you so much for being here with me today and sharing these tips about DNA purification and PCR and DNA cleanup. All super helpful. And I do want to point out that as Jack mentioned, he is on the tech support team, so you could actually get real life help from Jack with your purification needs, along with lots of other talented and knowledgeable tech support scientists that we have here. So just one more time, thanks so much for being here and sharing your knowledge with us today.
Jack Martz:
Thanks again for having us.
Heejin Kim:
Thank you.
Lydia Morrison:
Thank you for joining us for this episode of the Lessons from Lab and Life Podcast. Please check out our show's transcript for helpful links from today's conversation. And as always, we invite you to join us next episode when I'm joined by Nobel Laureate Dr. Carolyn Bertozzi, Dr. Bertozzi's impact on the field of glycosylation has been profound, enabling our current understanding of disease and inflammation presentation, and potential vehicles for treatment. You won't want to miss this insightful conversation.
