Ask the Experts: Everything you need to know about cloning
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Whether you are new to cloning or a seasoned expert, we have the webinar for you. NEB’s resident cloning experts provided an overview of various cloning methods, and answered common cloning questions received from customers.
Script
Deana Martin: Welcome to the NEBTV webinar series. Today, I am joined by three of our resident cloning experts: Lisa Maduzia, Yvette Luyten, and Rick Morgan. Hello everyone.
Lisa Maduzia: Hello.
Yvette Luyten: Hello.
Rick Morgan: Hello.
Deana Martin: And today, we are going to be answering all of your top cloning questions. We have a lot of questions ready to go, so let's get started.
Yvette Luyten: Good afternoon and thank you for joining us today for our Ask the Experts: Everything you need to know about cloning. My name is Yvette Luyten. I'm here with my co-workers:
Lisa Maduzia: Lisa Maduzia.
Rick Morgan: And Rick Morgan.
Yvette Luyten: Thank you so much, again, for joining us. We've received some great questions. But before we dive into the questions, we're going to give you just a little bit of background on cloning in general, so we're all on the same page.
Lisa Maduzia: Okay, so let's get started. So, what you can see up on the screen is a cloning workflow comparison. So, on the left hand side, you can see different ways that you could prepare your insert to get it ready to go into your vector. And we're going to go into further detail as we move along through the webinar. And then, on the right hand side is showing you how you can prep your vector to get ready it to accept your insert. This can be found on the cloning technical guide in the widget on the webinar console.
Lisa Maduzia: So, let's start with vector preparation. So, typically, you have a plasmid that you're starting with. And this plasmid would have a multiple cloning site, as you can see, shown here on the left. What you typically want to do is open up this vector so that you can get your insert in. And the way you can do that is you can use restriction enzymes. So, you can do a digest, and that would create either blunt ends, or you could use restriction enzymes that would allow for sticky ends to be created. Another way you can go about this is to do PCR off of a starting construct that you might have in the lab. And what's great about this is you could engineer restriction sites into your primers so that you could then continue on and cut. Or, if you don't want to go that route, and depending on what you're doing downstream, you don't have to have the restriction enzymes in.
Lisa Maduzia: So, once you get these opened up, you might want to do some DNA end modifications. And something that we suggest here, it's optional, dephosphorylate, but it's actually a really good idea. So, after cutting with your enzymes, you would then dephosphorylate. And what this is good for is, say, for example you have two enzymes you're cutting with, one doesn't maybe cut as well as the other. So, you get a lot of vector that is singly cut, basically. And so, it opens it up, and what you can do is dephosphorylate so that you don't get religating, religation happening with the vector back upon itself, creating a lot of background. So, by dephosphorylating, you prevent that from happening. And so, that reduces the background.
Lisa Maduzia: For PCR, a great thing to do, too, is to use the restriction enzyme DpnI. And this, DpnI actually cuts GATC, methylated GATC sites. And what it does is it eliminates background, as well. And it does this because it removes the background template that you put in, because DNA from E. coli is methylated. And so, it now can go and cleave that and reduce your background. So, that's really a good idea, as well.
Lisa Maduzia: Okay, so we were just talking about restriction enzyme digestion to prepare the vector, as well as doing PCR. And so, we kind of put a comparison together between these two and how you might want to go about this. Basically, they're both low cost. On both of them, you have many different vector choices that you could choose from. You have many different, for a restriction digestion, you have many different restriction enzyme choices. Because you have, it's a multicloning site, so you can choose how you might want to cut and paste everything in together.
Lisa Maduzia: With PCR, you have a choice of ends. You can design and put in whatever restriction sites you want to put in. You have the option to add these restriction sites in. And then, with restriction digestion, you have directional cloning, as well as with PCR you have directional cloning. So, you could put in your insert in a particular way. With restriction digestion, the availability of appropriate sites can often be limiting. So, you only have the multiple cloning sites. The restriction sites in the multiple cloning site. And it often requires dephosphorylation to avoid background, which is what we were just talking about in the previous slide.
Lisa Maduzia: With PCR, you can create vector ends at any position, because you can design your primers. So, anywhere in the vector. If you want to be near the multiple cloning site, or if you want to be somewhere else in the vector, you can start anywhere you want, because you're designing the primers that you're going to use to create your vector. And DpnI can be used to reduce the background, which is what we talked about in the previous slide, as well.
Yvette Luyten: Now, Lisa was just talking about the various methods for preparing your vector DNA to accept the inserts when you're cloning experiments. We're going to shift focus a little bit now, and talk about actually preparing your insert DNA. In what is typically thought of as traditional cloning, you can prepare your insert in one of two ways. You can either digest the DNA with a restriction enzyme, or you can PCR amplify a region of DNA, or your gene of interest.
Yvette Luyten: So, I'm first going to focus on the restriction digestion pathway, in which you're cutting either genomic DNA, plasmid DNA, or even PCR product with a restriction enzyme. Now, your restriction enzyme will leave you with a five prime overhang, a three prime overhang, or a blunt-end. And depending on the vector that you're going to go into, you may need to do some end preparation on that fragmented DNA. So, if, for example, you're going into a vector that's been prepared to accept, or prepared to be used as blunt-ended ligations, you're going to need to get rid of either the three prime or five prime overhangs that have been generated by some restriction enzymes.
Yvette Luyten: To do that, you're going to take your restriction enzyme digested DNA, and you're going to perform a blunting reaction. You can do that using either T4 DNA polymerase. The DNA Polymerase I Klenow large fragment or even a Mung Bean Nuclease. All of these are going to either fill in the five prime overhang or chew back the three prime overhang generated by the restriction enzymes.
Yvette Luyten: Once you have a nice, blunt insert, you then put that over a cleanup column, in general, to get rid of all of the enzymes. And you have your insert that's ready to feed into a ligation. On the other hand, if you've cut with a restriction enzyme, that is either going into a blunt-end, or you're going into a vector that's prepared for sticky-ended ligation, you don't need to worry about that blunting, side blunting reaction. And you can go directly from your restriction digestion onto the column purification kit to get rid of your restriction enzymes. And again, you're left with a DNA insert molecule that's ready to feed into your ligation reaction.
Yvette Luyten: The second method of preparation is by PCR amplifying either a region of DNA that you're interested in, or your gene of interest. As Lisa mentioned with the primers that you designed for your vector, you can also design restriction sites, or recognition sites, for specific restriction enzymes into the forward and reverse primer for your PCR amplification. Once you've got your PCR amplicon, you can then feed into the restriction digestion pathway where you digest your amplicon, and then clean that up, and you've got an inset with sticky-ends ready to feed into ligation. Or, in some cases, you really don't need to...
Yvette Luyten: In some cases, you're not really interested in adding restriction sites to the PCR product. For example, if you're going to do TA cloning. So, you've amplified your DNA with a Taq polymerase. And the Taq polymerase will leave A overhangs, and allow you to go straight into TA cloning. So, in that case, you're going to want to take your PCR product, simply clean it up with your column purification kit, and it's ready to go. If you, on the other hand, have used a high fidelity polymerase to amplify your gene of interest, that's going to leave blunt-ends. And it's setup nicely for blunt-ended ligation. However, the thing you will have to do, is you'll first cleanup your PCR reaction, to get rid of the polymerase and dNTPs. You're then going to what to phosphorylate your insert so that the ligase has some way of joining your insert to the vector. So, again, if you've dephosphorylated your vector, which we definitely recommend doing, especially for blunt-ended ligation, you need to add the phosphate groups onto your insert DNA because the polymerase itself does not.
Yvette Luyten: The next slide that we're showing is a screen shot of our NEBcloner web tool. And this is a really nice tool for not only people first starting cloning, but also, you old experts and people who are old hands at cloning. It really takes you through the full range of traditional cloning workflow, as you can see on the top. It goes from restriction enzyme digestion all the way through to transformation. The buttons underneath, you can see if you can click in anywhere along the workflow to get our best recommendations, as well as protocols specific to the application and the recommended product. So, check this out. Take a look at it. You can find it either on our webpage, under tools and resources, or you can find a link to it in the widgets on the webinar console.
Yvette Luyten: And the final thing about traditional cloning, we just want to give you a little bit of an overview of the advantages and potential disadvantages to traditional cloning. So, as you can see on the left side, it's a low cost, very versatile method for cloning your DNA. It provides you many different vector choices and also makes directional cloning super easy. One potential disadvantage is the fact that you have sequence constraints due to the presence of, or a lack thereof, of restriction sites in the vector. So, you're limited to what is in the multicloning site. Or, if you have a restriction enzyme site that you want to use, but it happens to occur within your insert, you then have to go searching for another restriction enzyme to use. So, that's one of the potential drawbacks. But again, in my book, traditional cloning is awesome.
Rick Morgan: So, let's take a look at seamless cloning. Here, we're going to cover two methods. One is HiFi assembly, which is also known as Gibson assembly. And the second is Golden Gate assembly. So, in HiFi assembly, we're going to generate vector and inserts that have unique overlapping sequence at each end. And then, in the assembly reaction, you assemble the DNAs together. And then an exonuclease chews back the five prime ends to generate a single strand of three prime overhangs that can then anneal to each other in the complementary fragments. And then, the DNA polymerase comes in and fills in the gaps between the ends that have annealed together of each fragment, bringing them together, and then a DNA ligase seals the nicks that are left from the polymerase to generate covalently closed and assembled DNA. And you can assemble multiple fragments with HiFi assembly. Two or three are typical. You can go even higher, 10 or more. And typically, you have one vector with a few insert fragments.
Rick Morgan: So, another seamless cloning method is known as the Golden Gate method. Here, it's a variation on restriction enzyme cloning that takes advantage of special type IIS restriction enzymes that cut to one side of the recognition motif. Typically, BsaI, BsmBI, or BbsI are used. And these leave a four-base non-defined overhang. So, in this method, you setup your inserts and vector with a design so that the restriction enzyme, BsaI or other, cuts itself off the end of each fragment and leaves a unique four-base overhang. Then, the fragments can be assembled based on the unique four-base overhangs going together between the different fragments, so that you can put the fragment, ligase the fragments together into your desired order to generate the construct you're trying to make.
Rick Morgan: So, we have some handy online tools to help with the seamless assembly. The NEBuilder tool is great for HiFi assembly. It'll walk you through the requirements and how to design primers for the fragments and create the fragments and vector for the assembly. So, then, another wicked easy tool for the Golden Gate assembly can be found on our website, the NEB Golden Gate. And it particularly helps in designing the assembly and in choosing how to create the unique four-base overhang sequences to assemble multiple fragments together. And I think our record for Golden Gate here, in house, is something like 20 or 22 fragments assembled together; which is more than typically needs to be done. But it allows quite a few fragments to go together.
Rick Morgan: So, looking at some of the advantages and considerations for seamless cloning, the nice thing is that there's no sequence constraints. Typically, you can put things together however you like. It's very efficient for assembling multiple fragments. You get high cloning efficiency and you get excellent control of the gene order as you put it together. And especially, with the HiFi, in particular, it ends up being wicked easy to create your constructs. Some of the cautions are that typically these require, you're using PCR to generate your fragments. So, if you have a sequence that's difficult to amplify, that's just a consideration. You can also use synthetic gene blocks for fragments. And with Golden Gate, another consideration is that there needs to not be restriction enzyme sites for the BsaI or BsmBI within the inserts or the vector. So, just make sure that those are not present to complicate things in the Golden Gate.
Yvette Luyten: We've now given you an overview of the various ways to prepare your vector DNA and your insert DNA for cloning, as well as a couple different methods that are typically used for constructing your plasmid of interest. What we're going to do now is talk about the troubleshooting tree that we use when addressing tech support questions involving failed cloning experiments. Oftentimes, we get these phone calls into tech support at the end of the whole cloning experiment. Because, somebody has not, either gotten very few colonies, or they've gotten no colonies on their plates. But as you can see here from the troubleshooting tree, there are a number of steps along the way from the start to finish where things could potentially go wrong.
Yvette Luyten: So, what we're going to do is try to give you a few pointers and things to look out for to help you, so you can have a successful cloning experience. You can also, in addition to what we talk about today, find a comprehensive troubleshooting guide. It's linked to, again, on the widgets on the webinar console. But if you also go to our webpage and type in the search bar cloning troubleshooting guide, that should also come up.
Rick Morgan: So, DNA purity is important and the isolation method used influences the purity and the size of the DNA fragments. There are many methods to make DNA, but whatever method is used, the cleaner the DNA, the better. So, there a number of potential contaminants that can end up in the final DNA. If you've used phenol in the prep, ethanol from columns, whatever's been used to open up bacteria. If you had Proteinase K or detergents, there could be salts, RNA left.
Rick Morgan: So, making the DNA as clean as possible is important. And then, also having a good idea of how much DNA is there. The quantification is also important. So, if there are contaminants such as ethanol left over or other things in the DNA, sometimes there might appear to be more DNA when you quantify them there is actually present. So, for example, some methods read fluorescence in interpreting DNA concentration. But if there's something else, like RNA or something, a contaminant that fluoresces, it can throw the DNA quantification off.
Rick Morgan: So, if you're having any issues, it's good to use multiple DNA quantification methods. So, it's always great to run a gel, so you can actually visualize the DNA, see the size and about how much is there. You could use a Qubit®, you can use Nanopore, but whatever method you use if you're having problems, try a couple of different methods. And if the methods don't agree on how much DNA is there, it's pretty likely that the DNA is not pure, there's something else present and the DNA might benefit from further cleanup, running it through a cleanup column or something like that.
Rick Morgan: So, considerations with the insert DNA preparation, if you've PCR amplified, the polymerase that you've used will have an effect on whether there are As on the end of the DNA or not. And a consideration is just to check the PCR to make sure you have the product that you want and not other non-specific primers, such as primer-dimers or other things. Because, if there's more than one PCR product you may need to prep the fragment that you want or the additional products might interfere. They might get into the cloning reaction and give you a construct that's not what you want.
Rick Morgan: If you're starting with genomic DNA, again, the quality of the DNA and whether there are contaminants in it are important to look at. And then, another consideration if you're doing a restriction digestion to create your inserts... It's good to know whether your DNA is methylated or not. And if so, if it is methylated, then whether the restriction enzymes you're using are sensitive to that. Sometimes if the starting DNA has a methylation that blocks the restriction enzymes, that can create issues.
Rick Morgan: Also, enzymes can be salt sensitive. So, it's good to have as pure of DNA going in as you can. Another consideration with restriction enzymes is if the recognition motif for the enzyme is very close to the end of the DNA. Say, right at the end. That can be an issue. You generally need to have a few base pairs, one to four for each enzyme beyond the recognition site. And then, if you're doing a restriction enzyme digestion, after following PCR, we do have a chart that shows activity of the restriction enzymes in polymerase buffers. But generally, it's probably better to cleanup the DNA from the PCR reaction and then go into the restriction enzyme cutting.
Yvette Luyten: The next step in the cloning protocol that warrants some consideration is the DNA purification method, whether it's how you're purifying your insert DNA or your vector DNA. So, typically, this is done in one of two ways. You can either do a gel purification or a column purification. If you are doing a gel purification, there are a few things you really need to take into consideration. The first is the fact that the UV light source that you're using can have a tremendous impact on the quality of your DNA and the actual outcome of your cloning experiment. We'll get into this in a little more detail in the next couple slides. But if you're going to be visualizing your DNA using a UV light source, you want to make sure you're using a long UV wave length or 365 nanometers.
Yvette Luyten: The other thing to consider when doing gel purification is you want to minimize the amount of agarose in the plug that you're cutting out. So, when you're cutting out your bands, try not to take these broad swaths of agarose along with your DNA. Something to also consider, and this is for both gel purification and column purification is that elution off the column the column from your, of your DNA molecules is going to vary depending on the size of the DNA molecule. So, larger fragment DNAs don't come off as readily. So, you really want to pre-warm your buffer, let it set on the column for one minute prior to spinning to elute the DNA. Finally, for gel purification, we recommend making sure that when you're dissolving the agarose plug you keep, the reaction, you keep the water bath below 60 degrees. Simply because once you start getting over 60 degrees it starts messing with your DNA, and again, can impact downstream in the number of tranformants that you get.
Yvette Luyten: Finally, looking at the column purification, one thing to really keep in mind is that although it's a purification technique, you will still have residual salts and a little bit of ethanol. So, this is where and why we recommend that when you're using DNA coming off of a column purification, no more than 25% of your total reaction volume should be accounted for by the amount of DNA you're adding. So, for example, if you have 100 microliter reaction, no more than 25 microliters of that should be your DNA. Because that allows residual salts or ethanols to really start messing with your enzymes.
Lisa Maduzia: Okay, so another area to look at, for when you're troubleshooting is to look at end treatment and vector preparation. We've talked a little bit about both of these. In the case of end treatment, you might be phosphorylating the ends or dephosphorylating the ends, you might be A-tailing for TA cloning, or blunting and filling in. With vector preparation, when you amplify your vector through PCR, restriction digesting is another area to focus on if you're having problems, gel prepping the vector, which we discussed some, as well, column prep could be an issue, or dephosphorylation of your vector. All of these things are something to think about in case you're having issues and you're using one of these methods, to look at. And you could also take a look at our... We have a detailed troubleshooting tips associated with each of these treatments, and these could be found on the widget of the webinar console.
Rick Morgan: So, an important consideration, if you're gel prepping, is whether you're generating UV damage to the DNA. Because high energy, which is short or medium wave UV light, does a lot of damage. So, there are three options you can use to get around this. One is to use a non-UV method for visualizing the DNA. There are dyes, like SYBR Safe that can be used. You could use a blue light box, if you have that in the lab. A second method is just to use a long wavelength, which is 365 nanometer UV light to visualize. And a lot of the UV light sources will have switches for different wavelengths. So, just check yours and make sure you're using long wave, which will not damage the DNA.
Rick Morgan: And then, another option if it's really important never to expose your DNA fragment, either the dyes, like ethidium bromide or SYBR Safe, or UV, is you can setup your gel, run it with the size standards and your insert of interest, and just cut out the size standards and part of your insert. And then, stain that, visualize that, mark where your fragment is, and then, cut the fragment out of the DNA that... out of the gel, sorry, that's never been exposed to UV light, or to a dye, and then prep the DNA from that gel slice. And that completely avoids any issues with UV light.
Rick Morgan: So, in considering the ligation reaction, one of the most important things is to remember that it's the number of molecules of DNA that matters, not the mass. And we have a useful tool for calculating molarity or concentration, which is the NEB Calculator tool. So, when you're ligating with your insert and your vector, if they are a similar size then you can use them roughly at a 1:1 ratio, if the insert is three to four times smaller than the vector, then you might want to go three insert to one vector ratio. And for smaller inserts still, increase the ratio of insert to vector size to drive the reaction to completion.
Rick Morgan: In terms of incubation time, four base overhangs are relatively easy to ligate. They can finish in 10 minutes at room temperature. If you have blunt or even single base pair overhangs, they're more difficult for the ligase to put together. So, we suggest a longer incubation time, maybe one hour to overnight. And also, to use the high concentration T4 DNA ligase or the Blunt/TA Ligation Master Mix to help drive the reaction to completion.
Rick Morgan: And if you're having issues with the ligation, there's a few controls that are useful to use. One is to end up transforming the cut and dephosphorylated vector without having ligase to tell if the vector has been linearized or not. You can cut, you can transform the cut and dephosphorylated vector after ligation to check for dephosphorylation. It should not go back together. If the dephosphorylation has worked. You can also cut the vector and ligate without having done dephosphorylation. And that tells you whether the ends are still intact. And then, it's also sometimes helpful to ligate just the insert alone, and look at it on a gel to see if you form a ladder, or larger pieces... It just tells you that the ends of the insert are ligatable. And they should go together to form a concatemer if everything is good.
Yvette Luyten: And the final step in the cloning protocols is, of course, your transformation. A couple things to keep in mind, it's important if you're having problems downstream to actually transform the control reactions Rick was just talking about for your ligations. So, again, your cut and dephosphorylated vector without ligation, the cut dephosphorylated vector with ligation, your cut and religated vector, to test that the ligase is working and that your ends are ligatable, and finally, to transform some of your undigested vector. This will tell you that, or confirm, that yes, you're using the right antibiotics, that the plasmid that you're using is what you think it is. There have been occasions where maybe that's not always the case.
Yvette Luyten: Other things to consider for transformation are the genotypes of the competent cells that you're using. So, you don't want to use a comp cell that's going to cut methylated DNA if you're transforming methylated DNA. The other thing to think about it potential gene toxicity. Plasmid size matters. So, if you're trying to transform larger plasmids, for example, 10kb or larger, we recommend using our 10-beta comp cells. And finally, if you're doing or using electrocompetent cells, be aware that PEG and salt have a major impact on the transformation using those types of cells.
Yvette Luyten: So, we want to thank you for your attention during this overview of cloning. And we've got a lot of great questions that we're going to get to now. So, feel free to submit your questions, and we'll start tackling them. Thank you.
Lisa Maduzia: Hi there everyone. So, we have a great question that came in from a professor asking about the top 10 tips that we could give on cloning to his first year graduate students. And we thought this was a great, great question, not only for new graduate students, but also to remind all of us how to go about cloning sometimes and what to think about when we're having problems.
Lisa Maduzia: So, one of the things also asked was, is there like some resources that we would recommend? And we really would recommend, as we spoke about in the actual webinar that your just were watching, we would really recommend the NEBcloner tool. And you can find that right on our website, on the first page under tools and resources. And that just takes you through. And that, you can go in there and play around and even when you have nothing to clone, just go in there, play around. It will help you figure out what protocols, what products are best, and for whatever application that you're trying to do, whether you're trying to cut or ligate into a vector or transform, et cetera.
Lisa Maduzia: So, some of the top 10 things that we came up with were purity and quality of the DNA has everything to do with the end result. So, you really have to know that your DNA is from a good source. It's prepped well. Anything, contaminants in there, can get in the way of how you're trying to, when you're trying to clone and get your construct made properly. We strongly recommend using a high fidelity polymerase such as Q5®® for PCR amplification of your genes just so you don't get any mistakes put in into your gene of interest that you're trying to clone into your vector. That's huge.
Lisa Maduzia: Another huge one is mass is not the same as molecules of DNA. And it totally matters. So, I think sometimes people forget that. So, we have a great tool called the NEBioCalculator tool. And that helps you calculate that so that you'll know if you have a... So, basically, if you have a large piece of DNA it has a larger mass, but it has less molecules in that same amount. So, you have to remember a smaller amount of DNA, a smaller insert has more molecules. So it does matter, mass versus the number of molecules. So, that's really important to think about. So, the NEBioCalculator helps with that.
Lisa Maduzia: The fourth thing we have on this list is to know the methylation status of your DNA. And the methylation sensitivity of your restriction enzymes. So, if for some reason you are trying to cut, for instance, we talked about DpnI, if you're trying to cut some DNA and you're not getting cutting, is it because your enzyme actually recognizes the cut site when it's methylated, and you do not have methylated DNA? So, keep that in mind.
Lisa Maduzia: E. coli methyltransferases. So, we have dam methylation, which is on GATC on the A. In that sequence, you got DCM methylation on CCWGG on the C site. And mammalian methyltransferases to be aware of are the GP... I mean, excuse me, the CpG methylation, where the C is getting methylated.
Lisa Maduzia: UV light. That's number six. UV light does a lot of damage to DNA. Using a long wavelength of light, such as 365 nanometers to visualize when gel prepping your DNA, that's really helpful. So, that way you're not incorporating any extra mutations into your DNA that you're trying to clone into your vector.
Lisa Maduzia: Do control reactions. That's huge. So, say you cut a plasmid. Cut, make sure your enzymes are working if you're having issues. Cut the plasmid, and then see if you can then actually religate it back and transform it into cells and you get that reaction working. So, you know all your enzymes are working. So, do controls. Controls are huge.
Lisa Maduzia: More is not better. So, too much restriction enzyme can cause off-site cutting. And too much template DNA can inhibit PCR amplication, if you're, especially if your DNA isn't clean, as we were talking about, with the quality and purity. Too much DNA in a ligation can also decrease your efficiency. So, be aware of those things.
Lisa Maduzia: Wow, we have number nine. We have familiarize yourself with the online tools. So, there are, as we told you, there are wicked awesome online tools. There's NEBcutter, there's NEBcloner, the NEBioCalculator, the Tm calculator to help you figure out what temperature you need to be using your primers at when you're doing your PCR reaction. So, that, that's huge, and those are really use and they're wicked easy.
Lisa Maduzia: And when you don't get transformants... Always, a lot of us here, we talk about a lot, we laugh about it. When you don't get transformants on your plate, everyone seems to go and blame the ligase, but it's almost never the fault of the ligase. So, you've kind of got to move on from that. So, that's, those are our top 10.
Lisa Maduzia: So, that's a great list, and hopefully that will help all of you go on and get your cloning done right. And in general, one last thing I want to say on this is to just follow our protocol guidelines. The protocols have been worked out by the scientists here, and if you follow them and don't stray from them, you will most likely get what you want cloned into your vector.
Yvette Luyten: All right. So, another question we received was regarding the addition of a His-tag to either the C terminus or end terminus of the protein. And the question was in case... "Does a spacer need to be added between the end of the gene and the His-tag, and if so, what would be the ideal length of the spacer?" And in answer to that question, for most proteins they're a linker or a... Yeah, a linker is not needed between either the end of the gene and the His-tag. Just make sure that you take out your stop
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