DIY method for isolating yeast

STORM, PALM And fPALM- The Alphabet Soup Of Super-Resolution Light Microscopy

Why do I see a cloud? When you are looking at protein localization within a cell, have you ever wondered why you see a cloud of fluorescence rather that several individual fluorescent points? Well, light microscopy has a theoretical resolution limit of 200 nm. This means that in theory, to resolve two points as being…

Ignore The Ticking Bomb At Your Peril: RNA-Seq Normalization- A Lurking Problem And It’s Solution.

You have spent days, if not weeks, at the bench setting up the treatment and control samples for that crucial experiment. You submitted your cDNA library for sequencing and after a few weeks of waiting anxiously you get back a list of differentially expressed genes. Hooray?! Hold on- not quite yet! There is something you…

RNases: Their baddie super-powers explained (and how you can defeat them)

RNases are like the baddie super-heroes amongst laboratory enzymes. They are omnipotent, destructive and seemingly indestructible. This is because they were created by evil overlords, for the sole purpose making life difficult for the brave scientists who battle every day to produce high quality, intact RNA preps. Ok, I’m joking about the overlords part.  But…

An image of colors to depict care for your pH meter.

A Beginners’ Guide to Non-coding Sequence Alignment

There is no such thing as “junk” DNA Until recently, vast areas of the genome had been denounced as “junk” DNA, because they do not encode proteins. However, it has become clear that these regions have a large diversity of other functions, from transcriptional and translational regulation to the protection of genes and genome integrity….

When NOT to Wear a Lab Coat and Gloves: A Quick Guide to Personal Protective Equipment

Personal Protective Equipment (PPE) such as your lab coat and gloves are an essential piece of kit when working in a laboratory; however, it is important to know when it is inappropriate to use PPE. 1)      Don’t wear your lab coat, gloves or other PPE in offices or dining areas. They may have become contaminated…

Top Tricks for Isolation of miRNAs from Plasma and Serum

MicroRNAs (miRNA) are short, non-coding RNAs involved in post-transcriptional silencing of gene expression. miRNAs can be associated with exosomes and can function as cancer-specific biomarkers. This, coupled with the fact that they are stable in plasma and serum makes them valuable diagnostic tools, as long as they can be reliably isolated from the serum and…

How to Screen for CpG Methylation by Methylation Specific PCR

How to Screen for CpG Methylation by Methylation Specific PCR

In a recent article, I gave some tips about how to obtain good results with sequencing DNA after bisulfite conversion (it contains some tips that apply to the approach described in this article, too). Bisulfite sequencing is a very useful technique if you want to know the methylation status of every CpG in your genomic…

6 ways to show your plasmid preps some TLC and get more supercoiled plasmid in return

In my last article, I explained that plasmid DNA recovered from a plasmid prep consists of few different species; supercoiled, nicked, linear and single stranded circular, and how you can distinguish them on a gel. Supercoiled DNA is the desired form of plasmid DNA; it performs better in downstream applications such as automated sequencing and…

Immunohistochemistry- Direct vs. Indirect Methods, and a Golden Rule

Immunohistochemistry- Direct vs. Indirect Methods, and a Golden Rule

In my previous article I covered different immunohistochemical staining techniques at a superficial level. In the following articles I will start to explain these technologies in a bit more detail and in which situations they should be applied. All of the following will involve additional stages when applying them, for example- serum blocking, protein blocking,…

Do Your Homework to Find Good Reference Genes

Do Your Homework to Find Good Reference Genes

Comparing and measuring gene expression is certainly an integral part of research—gene expression patterns continue to show us how different cell networks are regulated, and point to new biological pathways and possible treatments for disease. But one crucial part of gene expression lies in making sure that differences in gene expression are due to gene…

How Can A Single Mutation Affect Splicing Regulation?

How Can A Single Mutation Affect Splicing Regulation?

Alternative splicing is a highly orchestrated process that uses a multitude of regulatory mechanisms. Splicing specificity involves a precise interaction between cis- and trans-acting regulatory elements, and factors that disrupt these interactions can result in aberrant splicing. There are multiple ways in which mutations can affect splicing fidelity: Global analysis of alternative splicing is essential…

7 Tips for Preparing Chromatin for ChIP from Tissues (Rather than Cells)

A commonly used technique in epigenetics is Chromatin Immunoprecipitation, or ChIP for short. This technique can show you whether a certain protein (e.g. transcription factor or histone modification) binds to DNA, when in its native conformation, namely chromatin. Insightful, but difficult This information can be very insightful, but difficult to obtain. Most protocols and suggestions…

Second Chance Saloon: How to Western Blot a Coomassie-stained gel

Second Chance Saloon: How to Western Blot a Coomassie-stained gel

In her article How to Get Perfect Protein Transfer in Western Blotting, Emily Crow recommends Coomassie staining your gel after transfer to the membrane to check the quality of the transfer. A good transfer should not leave behind proteins and PVDF membrane, stained by 0.1% Ponceau S in  5% phosphoric acid and destained with water…

10 Ways to Improve Your Bisulfite Sequencing Results

10 Ways to Improve Your Bisulfite Sequencing Results

The importance of epigenetics in biology is increasingly acknowledged (if you’re not convinced yet, read my crash course). One commonly studied epigenetic mark is CpG methylation: cytosines that are directly followed by a guanine nucleotide (indicated by CpG), can be methylated, unlike non-CpG Cs. Since attachment of a methyl group to a cytosine can affect…

Three Tips (and Two Tricks) for Using BLAST

Three Tips (and Two Tricks) for Using BLAST

The Basic Local Alignment Search Tool (BLAST) algorithm is at the heart of a free suite of online resources available through the National Center for Biotechnology Information (NCBI).  While most researchers are aware of BLAST as a sequence alignment tool, NCBI’s BLAST suite offers so much more!  I’ll cover in-depth how to use these resources…

Immunohistochemistry- an Introduction, Techniques and an Evolution Towards Robots!

Immunohistochemistry- an Introduction, Techniques and an Evolution Towards Robots!

The last two decades have seen a dramatic increase in the number of publications using immunohistochemistry (IHC) as a research tool to identify the spatial location of proteins of interest within cells, tissue sections and whole-mount preparations. Grinding and binding The advantages over ‘grind and bind’ methods are apparent, but the very best results will…

Tips for Choosing and Using a New Primary Antibody

Tips for Choosing and Using a New Primary Antibody

Finding a good primary antibody can often feel like playing Russian roulette.  Nothing is more disappointing than buying a $300 antibody that doesn’t work for your use. There are some steps you can take, however, to increase your likelihood of success. Scout out Other Labs Before you buy, ask if anyone around you or in…

How to Detect Alternative Splicing Variants

How to Detect Alternative Splicing Variants

Alternative splicing events often occur in a spatiotemporal manner, and some are regulated by alternative splicing regulators, with striking variation across tissue types and developmental stages. Alternative splicing events are often differentially regulated across tissues and during development, as well as among individuals and populations, suggesting that individual isoforms may serve specific spatial or temporal…

Antibiotics and antimycotics in cell culture: how do they work and do I really need them?

Anyone who has spent any amount of time in a cell or tissue culture lab will have experienced contamination at some point. You might not admit it, or want to admit it, but you know you have! I performed my graduate work in a basement university lab with an out-of-service emergency exit door in the…

Restriction Enzyme Wars: The Natural Function Of Restriction Enzymes

Parents  of small children attending nursery know that the period of time from September to June is a succession of colds and flues for the whole family – children with their underdeveloped immune system exchange viruses, creating new potent strains. Well, that’s probably how bacteria feel all the time in the natural environment teeming with…