Get the most out of NEBuilder HiFi DNA Assembly
This webinar covers NEBuilder HiFi, a DNA assembly method by New England Biolabs, highlighting its efficiency and accuracy. The NEB product manager discusses the features, applications, and advantages of NEBuilder HiFi, including its ability to seamlessly assemble multiple DNA fragments using a simple master mix format. Detailed insights into the assembly process and troubleshooting tips are provided, making it a valuable resource for researchers looking to apply this method in their work.
Script
Welcome everyone. Thanks for joining today's webinar where we'll be talking about NEBuilder HiFi, an efficient DNA assembly method developed by New England BioLabs. My name's Ali Dolan and I'm a product manager in the Cloning Technologies group here at NEB. I'm joined here today by my colleague Lindsey, who is co-manager of the cloning product line, and Jack, our cloning support expert.
Here's a quick look at what we'll be discussing today. We'll start with an introduction to NEBuilder HiFi DNA assembly and go over how it works and why it's so useful. Next, we'll get into the features and applications. We'll walk you through what sets it apart as an exceptionally efficient and accurate assembly method and the main advantages it offers compared to other methods. Finally, we'll wrap up with some tips and tricks to help you get the most out of your experiments. That section will include troubleshooting strategies and a look at some of the tools that can make designing and setting up your assemblies even easier. Our goal is that by joining us today, you'll walk away feeling confident about how NEBuilder works and how you can apply it to your own research.
NEBuilder HiFi DNA Assembly Master Mix was developed here at NEB to improve the efficiency and accuracy of DNA assembly. Using this Master Mix, you're able to start with a linear vector and DNA inserts that have overlapping regions. After mixing, the reaction is incubated at 50 degrees Celsius for 15 to 60 minutes, leading to assembled DNA that can be directly used for downstream applications like restriction digest, colony, PCR, or sequencing.
This method can be used for routine cloning and also allows for seamless assembly of two to 12 DNA fragments with optimal assembly up to six using fragments as low as 120 base pairs and as large as 10 KB or more. The enzymes in the Master Mix work with high efficiency and accuracy, so you get the most of your assembled product as possible, meaning you won't need to screen as many colonies. It can be used to assemble either single stranded oligonucleotides into a plasmid or different sizes of DNA fragments with buried overlaps, ranging from 15 to 30 base pairs. And the end product is a covalently closed seamless DNA construct. NEBuilder HiFi is a great choice for one-step cloning of multiple fragments due to its ease of use, flexibility, and simple Master Mix format. In fact, it's the number one cited DNA assembly tool on the market with over 10,000 citations.
Now let's look in more detail at exactly what's happening in an NEBuilder HiFi DNA assembly reaction. Everything happens in a single tube isothermal reaction. The method works with DNA fragments that have overlapping complimentary ends. These overlaps can be added ahead of time, for example, by PCR. The fragments are combined with the Master Mix and incubated at 50 degrees Celsius for as little as 15 minutes or up to one hour, depending on the number of fragments being assembled. During the incubation, there are three enzyme activities in the mix that go to work. First, a 5' prime to 3' prime exonuclease trims back the DNA ends, generating single stranded 3' prime overhangs. These complimentary overhangs, then anneal aligning the fragments. A high fidelity DNA polymerase extends from three prime ends to fill in the gaps. And finally, a DNA ligand.
Now let's dive deeper into the advantages of NEBuilder and how it compares to other commonly used assembly methods. NEBuilder HiFi is the clear choice for efficient and accurate DNA assembly. It outperforms other methods in two, four, six, and 12 fragment assembly. As you can see here, NEBuilder yields far more colonies compared to GeneArt Gibson Assembly and In-Fusion Snap Assembly using both four and six fragments.
Another clear advantage of NEBuilder is its ability to remove mismatched sequences. Unlike other cloning methods, NEBuilder HiFi allows you to digest your DNA fragment with restriction enzymes and conveniently assemble it at any position. This is particularly helpful in scenarios where there are non-homologous sequences at the 3' prime M that are unwanted in the final assembly product.
When the exonuclease removes bases from the 5' prime end during fragment assembly, generating a 3' prime overhang with extra bases that aren't wanted in the assembly product, NEBuilder HiFi is capable of efficiently removing the 3' prime end mismatches, up to 10 base pairs, so that they can be efficiently joined together. Since the Master Mix removes any unwanted 5' prime and 3' prime mismatches, you can use NEBuilder HiFi in successive rounds of cloning, which can save you a lot of time performing extra PCR amplification steps.
Some of you might already be using other assembly methods like Gibson. And if you are using Gibson for your assemblies, switching is really easy. With NEBuilder HiFi, you get higher accuracy thanks to a high fidelity polymerase, meaning fewer errors and less screening. It's more efficient, giving you more colonies even from large fragments or low DNA inputs. It's flexible enough to fix 5' prime or 3' prime mismatches and versatile for bridging double-stranded DNA with synthetic oligos for rapid builds, which Lindsey will discuss in just a minute. Plus it's already compatible with your Gibson designs, and you can design primers easily with our free NEBuilder tool. So now I'll hand it off to my colleague Lindsey, who will go over in more detail some specific applications of NEBuilder HiFi.
Lindsey Spiegelman:
All right. Thank you, Ali, for that introduction and overview of typical NEBuilder assemblies. Now I'm going to change gears and take you guys through some alternative applications of NEBuilder that you may or may not be aware of so you can get the most out of your reagents. These are also great options if more traditional cloning approaches have not been working well for you. I'm going to start with talking about how NEBuilder can be used for single and multi-site directed mutagenesis.
Many of you are probably aware of site-directed mutagenesis kits. While these kits can certainly work well, they rely on multiple enzymatic reactions and tend to require colony screening to identify the correct clone. NEBuilder can also be used to generate a mutated construct as long as the mutation is introduced into your fragment before assembly. As shown here on the slide, this can be done by amplifying your fragment by PCR with the desired mutation contained in the five-prime primer, or ordering synthetic oligonucleotides containing the desired mutation and annealing them. After generating the modified insert, you simply proceed with your assembly reaction as usual, and your final product will contain your mutant with a typical high efficiency of an NEBuilder HiFi assembly.
One advantage of NEBuilder is that you're not limited to a single mutation at a time. The same principle can be used to introduce many mutations simply by breaking up your insert into multiple pieces with the mutations contained in the overlapping regions. In the example shown here on the bottom right, we show a final construct with three mutations. To achieve this, the insert fragment is amplified with four pairs of primers, placing the mutations at the overlaps. When input into the assembly reaction, the NEBuilder HiFi Master Mix assembles them all to generate a single product containing all three mutations. This is particularly useful when the mutations are far apart, for example, across different genes within the same plasmid. For mutations desired closer together annealed oligos can be used.
But what if you only want to insert a few dozen bases or create an entire library of mutants? Another unique feature of NEBuilder is that it can bridge two double-stranded fragments with a single-stranded oligonucleotide. This can even be done while simultaneously also assembling multiple double-stranded fragments. The single-stranded oligo bridge should have 25 to 30 bases of homology to the double-stranded fragments you want it to join. This can be done simply to combine two double-stranded fragments, or the oligo can contain a short insert sequence between the overlaps. During the assembly the T5-exonuclease will first chew back the 5' prime end of DNA fragment two shown on the right. This allows the single-stranded oligo to anneal to the second fragment and the polymerase to fill in the complimentary sequence, creating a double-stranded fragment. Then the exonuclease can chew back to the front end of the now double-stranded oligonucleotide, allowing it to anneal to DNA fragment one.
The polymerase will fill in any remaining gaps and lightly seals to mix. This method is especially useful if you have a short insert sequence because the exonuclease will not digest the ends of a single-stranded fragment. And that's not subject to the same length requirements as a double-stranded insert.
This method also simplifies library prep. If you need to create a large pool of mutants, such as those needed for CRISPR-based screening, it's much easier to use a pool of single-stranded oligos rather than generating thousands of unique double-stranded DNA fragments. You simply order your library of mutants as a pooled oligo collection, each containing the specific overlaps for the vector of your choice, and then use NEBuilder HiFi to assemble it with your plasmid and transform the assembly into high efficiency competent cells to generate clones with unique inserts.
And finally, I just want to briefly touch on the different NEBuilder products that we offer. Our standard NEBuilder kit comes with NEB five alpha competent cells suitable for most applications. But if you are assembling a particularly large construct, we also sell the Master Mix with our NEB 10 beta cells. And if you're all stocked up on competent cells, we do offer the Master Mix by itself.
If you want to learn more about the alternate applications I touched on today or need help getting started, we have detailed protocols and notes on our website on the NEBuilder product page. And now I'm going to turn things over to our technical support expert, Jack, who is going to take you through some challenges that our customers have faced with their assemblies so that your assemblies will hopefully go as smoothly as possible. Thank you so much.
Jack Martz:
Thanks, Lindsay. As Lindsay said, I'm Jack Martz. I'm the technical support scientist here at NEB that specializes in DNA assembly, as well as general cloning and protein expression. I'm going to briefly go over what the rest of today's talk is going to look like. I'll briefly do an overview of how technical support works at NEB, do a couple of case studies based on real customer cases that we've had about NEBuilder, go over some website resources that we have to make best use of your NEBuilder Master Mix, and then go over a final summary.
So here's a quick technical support overview on how technical support works at NEB. So NEB's technical support is for scientists by scientists. NEB's technical support is staffed by expert scientists. Several of our product lines have dedicated technical support scientists like cloning. Outside of this core group, most scientists at NEB also do technical support on a rotational basis. That includes product development scientists who actually have designed our products, research scientists who use our products every day for basic research, product managers who formulate and QC our products. And we're all driven by our core values, which are passion, humility, and being genuine. You can always reach out to us. Call us at 1-800-632-7799 Monday through Friday from 9:00 AM to 6:00 PM Eastern Time. And you can always dial 4, which is cloning, or 5, which is protein expression and glycobiology if you want to talk to me directly. You can also email us at info@neb.com or fill out a web form at neb.com.
So let's dive right into our first case study. So all of these case studies are loosely based on actual cases that we've had here on technical support. The names are different and a lot of the details are too, but the core concepts are the same.
So Peter called in. I gave my standard greeting, which is usually very similar from call to call. And Peter wanted to know if he could use our 5-alpha competent cells with NEBuilder. He can. We sell a bundle that includes both those cells and our Master Mix, so he can totally use both if he wants to, which confused him a little bit. His assembly wasn't working and he thought the cells might have been the problem.
So I wanted to take some time to get to know Peter's assembly pretty well. He was assembling two fragments, a gene and a tag, into a lentiviral vector for the first time. All of his fragments were generated by PCR and he was transforming into our 5-alpha competent cells. He didn't perform the positive control reaction that we include with the kit. And he only got a few colonies. When he screened them using colony PCR, they were background. So no fragments were present. I then asked Peter to perform the NEBuilder positive control to ensure the Master Mix was still working well. And I also recommended a diagnostic PCR to get a feel for where the assembly was going wrong. I'll go over both of those things on the next slide.
So my first tech tip is perform the positive control. NEB supplies positive controls with a lot of our different reagents to make sure they work well in your hands and make sure nothing has happened during shipping. If you've ever called technical support, you've probably been asked the question, "Have you done the positive control?" It's one of the first questions we ask. The positive control for NEBuilder is two fragments that assemble together, a linearized pUC19 vector backbone and a full length lacZ. Both are validated to assemble together well and transform well. It's the first step of troubleshooting to make sure the Master Mix is working as expected. You can also use blue-white screening with this positive control because of that full length lacZ.
So my next tip is to decouple your workflow. This is really to determine where the problem is. Usually for NEBuilder, the final outputs are at the very end. So that looks like number of colonies that you get on a plate, colony PCR if you want to do so, or saying your sequencing results. There's no information per se if it's an assembly issue or a transformation issue with the cells. Instead, we want to decouple these two different steps. So we want to know if the assembly is working and the transformation separately. To do so, we can use PCR. You can determine if the assembly works in vitro by amplifying your final product directly from the assembly reaction. If you want to know the protocol to do so, you can reach out. We'll happily give it to you.
Back to our first case study, which is Peter's assembly. Peter performed the positive control reaction and the diagnostic PCR that I asked him to. When he performed the positive control reaction and transformed it into our competent cells, he got hundreds of colonies using the recommended transformation protocol. That's a great sign. That indicates that the Master Mix is working really well and that the cells are still competent and viable. He was also able to amplify the correct size product out of the assembly reaction, indicating that this is not an issue with the Master Mix assembling his particular construct.
The next steps are that I asked Peter to transform his assembly into NEB Stable, a cell line for toxic, repetitive, or unstable constructs. This was because I thought that it might be a transformation issue because the assembly appeared to be working really well. And I knew that he was using a lentiviral vector, which can sometimes be a bit of a hard cloning case. I recommended he perform all growth steps, so that includes outgrowth and recovery, plate culture, and liquid culture at 30 degrees or 25 degrees.
That brings me to my next tech tip. Toxicity happens and it's way more common than you think. There have been... [inaudible 00:17:36]. Going over why toxicity might happen with DNA and protein constructs, but it's still a little bit of a black box. But it's still a little bit... Black box. It's predicted to arise when novel constructs place a metabolic burden on the cells and it results in no colonies on a plate, truncated products, odd indels, or insertions of the E. Coli genome. Toxicity unfortunately often happens unexpectedly. It arises often when folks are combining otherwise well-behaved constructs, so a vector that they've used many times before and an insert that has been cloned before.
The thing we recommend is to change the growth conditions, then the plasmid sequence. We want you to get the construct that you set out to get. So use a strain for toxic or large constructs first. Grow it at a lower temperature and then by using a lower copy number origin of replication or truncating the plasmid.
After all the troubleshooting, Peter finally had some success. Upon transformation of his construct into NEB stable cells and growth at 30 degrees, Peter got his correct clone verified by Sanger sequencing. What's likely is that the inserts that he was putting into his lentiviral vector placed a high metabolic load onto the E. Coli, so they excised them from the plasmid in order to maintain their antibiotic resistance and survive antibiotic selection while mitigating the toxic effects of whatever DNA sequence they didn't like.
That brings me to my next case study, which is Lex's assembly. I wasn't joking when I say that I answered the phone the same way pretty much every time. Lex was having some trouble with their NEBuilder assembly. It just wasn't working. They gave me a little bit of a rundown of their assembly and it was kind of complicated. They were doing a 5 fragment assembly with a variety of different fragments.
Let's go over Lex's assembly a little bit. Lex was assembling four different inserts into a vector. Three meant for synthesized gene blocks. One to PCR product, and the vector double digested by two different restriction enzymes. Cell one and BamH1, both high fidelity. And they gel purified the vector after digesting it. The gene blocks already had overlaps built in when they ordered them from their company. Assembly, but it was their first with this many fragments. The amount of DNA, molar ratios, and overlap length were all well within our recommendations. They were transforming into NEB Stable. The concentration of their vector, however, was quite low. So they had to increase the volume of their assembly to 30 microliters. Not the end of the world, but something to keep note of. The steps I had them do were to do a diagnostic PCR reaction to determine if it was an assembly or transformation that was failing. I also asked them to perform the positive control reaction, including the kit and a transformation control.
When Lex got back to me about amplifying their assembly out of the Master Mix, they weren't able to do so. This indicates either a PCR failure during the diagnostic or an assembly failure. We usually don't recommend that folks spend a significant amount of time optimizing this PCR just because it's just not a good use of folks' time most of the time. The positive control reaction that they did yielded over 200 colonies and their transformation control worked well. So I wasn't worried about the Master Mix failing. The cells were viable and competent. This indicates that their assembly isn't working for some reason.
I began to think about their fragment quality. During further discussion about their assembly, two more points came to light. They were using gene blocks straight in the reaction, so just reconstituting them and then using them. And their gel purified vector backbone was not only very low concentration, but had a very, very low 260/230 ratio indicating some sort of contamination. My next steps were to ask Lex to prepare their vector by PCR amplification to avoid gel extraction and to amplify their gene blocks prior to assembly.
That brings me to my next tech tip, which is to amplify your gene blocks. When assemblies with gene blocks don't work, amplify. Occasionally, for reasons that we don't really understand, synthetic double-stranded DNA fragments may not assemble well when used directly in the assembly reaction. We're not sure of the mechanism for this. When troubleshooting a reaction that's failing, one of the first things we do recommend is to amplify the synthetic DNA fragments by PCR. For some reason, that fixes the problem.
That brings me to my final tech tip. Trash in, trash out. So make sure your DNA fragments are squeaky clean. The success of NEBuilder depends on the quality of the fragments used. When purifying fragments, you need to keep an eye on measures of quality like concentration, 260/280 ratios and 260/230 ratios. NEBuilder is inhibited by guanodinium salts, which are common contaminants from gel purifications. Guanodinium salts will absorb at about 220 or 230 nanometers. So you'll be able to see it on the spectrophotometer. Avoiding gel extraction is best, but if you desperately need to do a gel extraction, we can give tips to help get your DNAs clean. That might look like using a Monarch Cleanup Kit, which we can absolutely help you do. This figure is from one of the app notes that we have here at NEB where one of our scientists went over the common contaminants and common problems that you see when using spectrophotometers. So with those tips, Lex was finally able to see some success.
Lex's newly amplified fragments were able to be assembled. With more complex assemblies, fragment quality becomes paramount. What might not be an issue for a two-fragment assembly will definitely become an issue for a more complicated six-fragment assembly.
I'm next going to go over some of the tools that we have to get the most use out of your NEBuilder Master Mix. The first tool that I want to go over is our NEBuilder Assembly Tool, which can help you design your assemblies. You can paste your sequence into the tools, decide how you want to create it, either restriction digest, PCR amplification, or a synthesized gene block. And then the tool will design primers to help you append homology regions for the assembly to work. You can also add spacers if you'd like and copy and paste the final assembly to take a look at. If you need help using this tool, you can always call. We're happy to help. We also have a really useful help guide with pictures in the tool that you can look through.
The next tool that we have is a newer one. It's our Protocol Calculator. This makes your life super simple and prevents you from having to do pretty much any math during your assembly. You can put in the length of your fragments and how concentrated they are after you've created them, and the tool will do all the math and tell you how much to pipette into your Master Mix. It really makes using NEBuilder super simple. And again, if you have any other questions, feel free to call in.
In summary, these are all of the tech tips that I recommend if you want to get the most out of your NEBuilder Master Mix. If you do all of these tips, you solve most of the problems that I see doing technical support on this product every single day. They are perform the positive control, decouple your workflow, toxicity happens, amplify your gene blocks, and trash in, trash out. As a reminder, you can always reach out to technical support for help if you need it. You can call us at 1-800-632-7799 Monday through Friday, 9:00 to 6:00. You can dial 4 or 5 to talk to me directly. You can also email us at info@neb.com or fill out a form on our website. We're happy to help you or answer any questions you may have about NEBuilder or any other product that we have.
Lindsey Spiegelman:
Hi everyone. Thank you so much for being here. This is Lindsey and I'm going to kick off our Q&A with a few questions that we got during the talk. The first question that I'm going to address is a common one and it's how many fragments can I assemble at a time maximum? This is a really common question. And so what we say is that we expect the vast majority of our customers to be able to assemble up to six fragments in a single reaction. You have a bunch of enzymes working simultaneously and there is a degree of random chance of getting all of these fragments to align and then get sealed together. So if you think about that, it's going to decrease in efficiency the more fragments that you have. We have successfully assembled up to 12 fragments, but this requires a lot of optimization to make sure that the overlaps and the purity of the fragments are really as good as they can possibly be. So just to repeat, we typically say up to six. With additional optimization, you may be able to do a few more.
The next question that I have is, can I use two oligos in an assembly? You can assemble two ssDNA with double-stranded fragments. They just can't be adjacent to each other. So you need to have double-stranded fragments in between the two oligos.
Really quickly, I just wanted to mention something that I just remembered about assembling more double-stranded fragments. So if you need to assemble more fragments, you can always do a sequential assembly. So this would involve, say, assembling four fragments and then using PCR to amplify the large product out of that assembly. And then you can use that as a single fragment going forward into a downstream assembly. I hope that helps if you have a lot of fragments to assemble.
And let's see, the next question that I have is, do you have any tips to reduce background colonies? So one of the really nice things about the enzymes in NEBuilder is that it's not going to re-ligate a vector. It will feel mixed from overlaps, but if you put in, say, a single-cut vector, it's not going to re-ligate that. So the only time we see background colonies with NEBuilder is when you have contamination of the plasmid from upstream prep. So it depends on how you're preparing your plasmid. If you are amplifying your plasmid by PCR, which is what we recommend, you can treat it with Dpn1 to remove that template vector. If you're doing a digestion, you can always optimize the restriction digest, do a longer digest, sequential digest, or do a gel purification. We generally recommend that you avoid doing a gel purification if possible, as Jack mentioned, because the salts from that can inhibit the reaction, but sometimes you have to. So if you can amplify by PCR, that would be the best way to improve your efficiency.
I hope that was helpful. And now I'm going to hand this over to Ali to answer a couple more questions.
Ali Dolan:
Hi, everyone. Couple more questions here. First one is, can you use NEBuilder HiFi to assemble a linear molecule? And the answer is yes. You can mix your fragments together with the Master Mix, and as long as you have sufficient overlap, you can assemble fragments into a linear product for downstream applications.
All right, another question here. There's someone who is using Gibson for assembling three fragments, but the efficiency is low. And they're wondering if switching to NEBuilder will improve the efficiency. Yes. With NEBuilder, you can expect more colonies with less background. And they're also wondering if they could receive a free sample to try it out. Yeah, you can reach out to our tech support team to request a sample, or you can go to nebuilderhifi.com and there's a form there you can fill out to request a sample. I'm going to hand it over to Jack who has a few more questions to answer.
Jack Martz:
Howdy, y'all. So I have three questions that I'm going to answer. The first is, why not just use NEB Stable cells for all of HiFi assembly? You totally can if you want to. This is mostly up to personal preference. If you just want to put everything in stable so that you don't have to worry about any sort of toxic effects of a construct, go ahead and do it. Some folks prefer to use 5-alphas or 10-betas, and that's okay too. It's really up to you.
The second question that I have is someone has a double-digested vector. They're wondering if there's any special consideration for PCR amplifying the insert. And the only thing that I would keep in mind is any sort of mutations that might happen during amplification. We'd recommend that you amplify your vector by high-fidelity polymerase like Q5 and sequence it after assembly. I would also keep in mind difficult sequences like repeats and high GC content and any secondary structure that could be formed by the fragments that can cause issues during amplification.
The third question I have is how long can the oligo bridge be when using ssDNA for an NEBuilder assembly? There's really no upper limit besides what can be synthesized by your favorite oligosynthesis supplier. It's just that we want to keep in mind secondary structure of the oligo, which can inhibit the polymerase in the reaction, in the NEBuilder reaction. So you want to not have any secondary structure in theory in that oligo. The longer an oligo is, the more likely that's going to happen.
Speaker 4:
Thanks so much Jack and Ali and Lindsay for answering all of those questions today. If we didn't have a chance to get to your question, we will be sure to follow up with you offline. And if you have any further questions or would like to reach out to us, please feel free to do so at webinars@neb.com or of course our technical support. Thank you all again and have a wonderful day.
