Join Us
Sign up for our feature-packed newsletter today to ensure you get the latest expert help and advice to level up your lab work.
When restrictions come in the form of paperwork and approvals, we detest them. Whereas, when the restrictions come in the form of enzymes, we love them, don’t we? Restriction enzymes play a key role in biotechnology research. Read ahead for six useful facts about restriction enzymes. 1. Restriction enzymes are helpful to bacteria Restriction enzymes…
You had been happily measuring your DNA samples with your NanoDrop for a while, until… you noticed that your results are not reproducible *gulp*.
Read on – here are 10 points that will help you troubleshoot the issue.
You have a new plasmid, now what do you do? You are excited to go further with your project. But before you can move on, you have to confirm the presence of your insert as well as the sequence and orientation of the insert. Is the insert the right size? Most people use restriction enzymes…
You open the incubator in the morning and to your dismay there are a hundred glorious colonies… on your vector-only control plate. While there are a number of potential causes, I’ll highlight a few of the more likely culprits and their solutions.
When I began a master’s program in 2008, the lab task I hated more than anything was running agarose gels for DNA. Something so simple, ubiquitous, and necessary gobbled up more hours in the lab than I care to remember. Even though we added the DNA stain directly to the molten agarose and didn’t have…
Some viral vectors are the little black dresses of cloning and expression experiments: They work for almost any occasion and always give you the results you were hoping for. Other vectors are more like ballgowns that only come out of storage for special occasions. Let’s wade through all the information out there and take a…
Phenol-chloroform extractions and ethanol precipitations can be a royal pain, but here are some tips to help you get tip-top results!
While they may not be as in demand as when they were the basis of sequencing projects, bacterial artificial chromosomes (BACs) are still used for a wide variety of projects. Based off of the F origin of replication, BAC vectors can stably maintain up to 300 kb of sequence in a single plasmid, lending themselves…
What is cDNA? …and how do you choose the right tissue to make a cDNA for isolating your gene of interest? Here’s what, and how.
Most of us use pretty standard transformation protocols for E.coli. Yours probably goes something like this: – Thaw the competent cells on ice – Add DNA – Electroporate (or incubate then heat shock for chemically competent cells) – Add rich medium (LB or SOC) – Incubate at 37°C (or appropriate temperature) for 30-60 minutes –…
Traditionally, if you’re hoping to clone a DNA/RNA fragment (or insert) into a vector, such as a BAC you would need: Expensive exonucleases, called restriction enzymes: pacman-like enzymes that chomp at specific sequences in your destination vector or fragments to be inserted (often just “inserts”). Sequence homologies between your inserts and your destination vector, called…
Plasmid incompatibility could ruin your experiments before you’ve even started. And if you don’t know what it is, you can’t troubleshoot it! This article explains plasmid incompatibility, so you can’t be caught out by it.
One of the most annoying traits of “classical cloning” is an imperfect system of discriminating between the clones containing an empty vector and vector with insert after cloning. Even when your self-ligation control plate is empty, you can have a lot of colonies containing an empty vector on the “vector + insert” plate. Even the blue-white…
When I buy a new sweater, I love finding out that it goes with several pairs of pants, the scarf that’s an awkward color and the earrings I haven’t worn yet. PCR is like this sweater – it goes with almost everything and molecular biology is taking full advantage of this using it at every…
I often wonder why it is that molecular biology researchers stubbornly refuse to change 40-year old methods that, while work, are not as good as newer, faster and cheaper methods out there. I suppose rational scientists often have irrational superstitions. One example of an old method that could be improved is the growth media used…
SLIC, or sequence and ligase independent cloning, was developed by Li in 2007 and published in Nature Methods. What makes it a Nature Methods worthy protocol? Unlike other forms of cloning, SLIC does not require restriction enzymes or a ligase! Seriously! Don’t believe me? Why not have a go for yourself? I’ve detailed the main steps below to get you started. How it works To…
Oligonucleotides are those smallish bits of DNA or RNA that we rely so heavily on for many of our molecular biology experiments. In their naked form, they are single, inert strands of DNA or RNA bases. But if you dress them up, you can increase their functionality. Here are some of the common oligo wardrobe…
Consider a jigsaw puzzle. While most of the pieces have a different picture on their surface, all pieces fit together in an interlocking pattern. As unlikely as it may seem, restriction enzymes from different organisms can produce interlocking pieces of DNA – so called compatible cohesive ends (CCE). These are pieces of DNA, which fit…
Look Ma, No Kit! Unlike with kits, there are no propriety reagents in a DIY protocol. You know what exactly is in each reagent. The DIY approach is catching on!
If efficient cloning is what you are after, you must give Overlap Extension PCR Cloning a go! This restriction enzyme and T4 DNA ligase-free technique is faster, more reliable and easier to troubleshoot than traditional restriction methods. With only two PCR reactions required, you can insert a DNA fragment into a plasmid without spending time…
If you ever used a site-directed mutagenesis kit or ligation-independent cloning, then you also used restriction enzyme Dpn I. But what does it do and more interestingly, why? Restriction enzymes and methylases: the yin and yang of bacteria Usual restriction enzymes, the toolkit of genetic engineering, are one half of the “yin and yang” pair…
Good news lab workers! Always hated the tedious work of designing a cloning strategy? Or maybe always dreamed of pooling all the reactions in one tube, just to save time? Thanks to Mother Nature, and her wonderful type IIS endonucleases, this is now possible! What is this wonderful enzyme? Type II enzymes are one of…
As frustration goes, cloning is often up there with trying to thread a camel through the eye of a needle. You do everything carefully: prepare your vector and fragment DNA, cut them as the restriction enzyme manufacturer instructs (complete digest in five minutes!), ligate, transform and go home in anticipation of a good number of…
While the classic approach to molecular cloning – using restriction enzymes to excise a DNA fragment of interest – is as useful as ever, new techniques that make cloning faster, easier and more versatile are available. As a smart molecular biologist, you should be examining each of them to see whether or not adding them…
They say a magician is never supposed to tell the secret behind his tricks. It’s a good thing then that I am a molecular biologist and not an illusionist. Because once I learn a good trick, I feel the need to spread it like dandelion seeds floating on a breeze. That’s what inspired me to…
Bitesize Bio has had a lot to say about RNA isolation, mainly because it is one of the most anxiety-producing requirements for molecular biology; especially when you are first starting out (although isolating proteins from complex samples like soil and stool is far more difficult, let me tell you. But that’s a future post.) We’ve…
As widely used as it is, isolating RNA remains one of the more finicky protocols. Just about anyone who has performed the technique has their own personal tips and tricks to successfully isolate intact RNA from their samples with consistency. Although RNA can be somewhat unpredictable since it is so labile, there are a few…
Sometimes a clever little trick for cloning comes along that makes you just give an appreciatory “ahhh.” For me, it was TA cloning. TA cloning is not a new technique (I am showing my advanced laboratory age here), but when I discovered it, the simple elegance of the technique made me pause and wonder about…
Every hard-core biologist knows designing the perfect construct can be a complex puzzle to solve. This challenge, if successful, can be extremely satisfying but can also drive you crazy for weeks. Luckily, Dr. Daniel Gibson and his colleagues at the J. Craig Venter Institute designed a new easy-to-use cloning method. Better yet, this system allows…
Blunt-end cloning involves the ligation of DNA fragments – usually between a plasmid vector and an insert – whose terminal ends are not “sticky”. Performing these ligations is notoriously difficult, particularly with large DNA fragments. But it is possible. And in this article I’ll give you some tips that I hope will increase your chances…
If you’re picking up a list of available plasmid vectors for the first time, it can be mind-blowing to decide on the best one for your experiment. What cloning sites do you need? What restriction enzyme sites? What insert size is possible? Let me help you with some pointers about what factors to look out…
Removing proteins from acqueous solutions can be a pain if you have loads of samples to do. This article explains how to prepare the DIY phase separating gel to easily and effectively separate two-phase mixtures, saving you time and money.
Plasmid mapping and DNA annotation software is pretty abundant these days. A quick Google search brings up dozens of hits – but how do you know which one to use? If you are like most molecular biologists, you probably use the same software your colleagues do—usually it is either the stuff that gets passed down…
According to the central dogma of molecular biology, DNA is transcribed into RNA, that is translated to proteins. Inconveniently, the vast majority of the genome contains sequences that do not actually code for proteins. So, this non-coding RNA (ncRNA) was dismissed as non-functional junk, letting researchers tick the box on their to-do lists and head off…
Recently BsB author Yevgeniy Grigoryev shared a total RNA isolation protocol. The one I use is even simpler—no expensive Trizol, which is a mix of phenol and some salts, all that is required is some Tris, SDS and phenol/chloroform mix. I have never used this protocol on non-yeast cells but I am almost sure that…
As part of my job ensuring plasmid quality at Addgene, I analyze 50-100 sequencing reactions a week. So I have developed some good habits that I wanted to pass on to you to make sure you are getting the most out of the data you get back from your sequencing runs. The most important of…
Unlike DNA, which can last for eons, RNA is a fragile and degradation-prone cousin. After working with RNA for a while, one becomes quite paranoid about handling RNA because even a single sneeze or drop of saliva can potentially affect your results. The reason is that there are enzymes called RNases that specifically target and…
Did you ever encounter resistance from a mammalian cell line when trying to extract the contents? Probably not, because destroying cell membranes is easy. Cell walls, however, are a different story. They are rigid, protective layers that can be so strong that the organism gives up movement in favor of protection! Cell walls exist in…
In my previous article on DNA gel extraction, I explained how most commercially available DNA gel extraction kits work. However, there was a time before our society was blessed with these convenient marvels of technology and scientists had to summon the gods of “Crush and Soak”. This method has been proven for millennia, as people…
Isolating pure DNA is key to many downstream applications for molecular biologists. Isolating large quantities of pure DNA used to be a laborious task. But thanks to commercially available kits, older methods have been streamlined to allow efficient recovery of pure DNA. In this article, I will talk about a method called DNA gel extraction,…
The eBook with top tips from our Researcher community.