DNA / RNA Manipulation and Analysis

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 –…

Welcome to the last article in the series on E. coli origins of replication where we will touch upon plasmid incompatibility! This is a broad and complicated topic so we will mainly focus on points that might be relevant for you in the lab. What is Plasmid Incompatibility? Plasmid incompatibility is usually defined as the…

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 4o-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 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…

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…

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…

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…

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…