Strengths and Limitations of the Amazing NanoDrop™ Spectrophotometer
With the NanoDrop spectrophotometer, quantifying a DNA, RNA, or protein sample concentration is as easy. Here’s everything you need to know about the strengths and limitations of this handy spec.
How DNA Extraction Kits Work in the Lab
Understanding how DNA extraction kits work is the key to troubleshooting your extraction issues.
Top Tips for Troubleshooting In Vitro Transcription
The Phobia of RNases My first experience of troubleshooting in vitro transcription came when I was synthesizing RNA In-Situ Probes for the first time. A lab mate ominously warned me that I had just returned to lab after a bout of flu and that meant I’m a walking talking factory of RNases. I went ahead…
Faster Ligations: PEGing down the Secret
Overnight ligations are inconvenient — especially when they fail. Luckily, there’s a straightforward way to faster DNA ligations. This article highlights the secret ingredient to faster ligation reactions and offers some tips and caveats on its use. For a general overview of DNA ligations, see here and here. Buy a Quick Ligation Kit The most…
CRISPR Genome Editing: What You Need to Know to Get Started
Get some ideas on what CRISPR can do for you and what using it involves.
How to Light Up your Life – Tips and Tricks to Troubleshoot your Luciferase Assay.
What is a luciferase assay and what is it useful for? A luciferase assay takes advantage of the innate bioluminescent properties some organisms exhibit, most notably the firefly. The firefly can convert luciferin to oxyluciferin in the presence of the enzyme luciferase to emit light. The most common scientific assays utilizing luciferase are reporter assays…
Three Important Things to Check After Obtaining Your Plasmid
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…
Faster, Even Cooler DNA Gels!
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…
Comparing Viral Vector Expression Systems
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…
Quick reference: Determining DNA Concentration & Purity
The most comprehensive way to evaluate DNA concentration and purity is to use both UV spectrophotometeric measurements and agarose gel eletrophoresis. This quick reference guide gives an overview of the information that can be derived from both. UV spectrophotometric measurement of DNA concentration and purity DNA itself, and most of the common contaminants found in…
Burning bright: a brief history of ethidium bromide DNA staining
For several decades, Ethidium Bromide (EtBr) was the molecular biologist’s default dye for DNA staining. Now, EtBr is being consigned to the history books. It’s time to have a historical look at where it all started.
What’s The Problem With Ampicillin Selection?
Ever wonder what those small colonies, like satellites, surrounding a larger E. coli colony on your LB with ampicillin plates were? Or why, when you picked that colony, it never had the plasmid you just transformed? Well, it’s because those satellite colonies are “protected” from the ampicillin by the big colony. Read on for more… Ampicillin…
Southern (blot) exposure remains a useful technique
At a meeting recently, I asked two PhD molecular biologists about the last time they used a Southern blot. After nearly a minute of unrestrained laughter, they asked “Who on earth still does that?” “Maybe for a very, very specific use,” conjectured one of the scientists. When I asked the scientist who taught me the…
Get Ready, Get Set, Retro – How to Get Started With Retroviral Transduction
Retroviral transduction is becoming a popular choice for gene delivery into mammalian cells and has multiple advantages over other techniques. If you decide to start work on this useful technique, here is how you can go about it: Step 0: Obtain permission First and foremost, do you have the permission, authorization, and training to work…
How to Cheat QuikChange™
Generating a DNA vector library of single and/or compound mutants for a target protein can be a daunting task. If you’re lucky and work in a well-funded lab, you might outsource this process via gene synthesis. Most of us though, need to do it the old-fashioned way. Traditional QuikChange™ Traditionally, there are many steps you…
Retroviruses – Friends or Foes?
The word ‘retrovirus’ evokes images of HIV, the AIDS pandemic and a desperate worldwide effort to defeat this mighty adversary. But on the flip side, scientific research has managed to tame the virus and use it as a tool for advancement. Retroviruses are commonly used to introduce genes into mammalian cells to express or knockdown…
4 Tips for Extracting RNA from Unfriendly Tissues
Some tissues are tricky to work with. This truth was lost on me in the early years of grad school because I worked with liver samples. If you’re extracting RNA from liver samples, you’re likely not losing sleep over your massive RNA yields. But for the folks doing RNA extractions with less willing donors, such…
Six Facts About Restriction Enzymes
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…
How to Fix Your Bad Cloning Ratios
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.
More Than a Clever Name: Northern Blots
You might think Northern Blots are an old-fashioned technique. However, qRT-PCR is prone to false positives and negatives, and reviewers may require Northern Blot confirmation of your qRT-PCR results. So sometimes Northern Blots are a necessary evil.
Defeat RNAse Contamination Using Bleach in Your RNA Agarose Gel
So, you’ve extracted your precious RNA and want to check its quality on a gel. Conventionally, you would run a formaldehyde gel, which is messy and requires a lot of prep. Plus, it is a huge undertaking in terms of time (and money) if all you want to do is just check the quality of…
Improving Library Preparation for Next Generation Sequencing
Library quantity and quality are paramount to the success of a Next Generation Sequencing (NGS) experiment. But there are a lot of factors that impact library yield and quality: conversion rate, GC coverage, low quality or low quantity input material, and PCR-introduced bias. All that, and wouldn’t a shorter protocol be nice too? Whether you…
-
Baby Got BAC – Working with Bacterial Artificial Chromosomes
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…
Cloning Round Up
Cloning can be the bane of a scientist’s existence. Rarely is cloning the end goal of an experiment. Generally it is the means to an end for studying something interesting that piece of DNA does, such as expressing a protein. That’s where we come in. We have tips and tricks for all sorts of cloning.…
Seamless Ligation Cloning Extract (SLiCE) Explored and Explained
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…
Banish the Background with Toxin–antitoxin Cloning Systems
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…
Simple Tips to Improve your Cloning Efficiency
Since the early 1970s, molecular cloning has used restriction enzymes to excise fragments from source DNA and to linearize plasmid vectors. After purifying the insert and vector, and ensuring compatible ends, both are joined with the help of a DNA ligase. The resulting recombinant vector is then used to transform an E. coli host into…
Choosing the right plasmid vector: A Guide for beginners
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…
DIY Phase Separating Gel: Clean and Cheap!
Learn from my adventures, save yourself some money and enjoy the nice phase separations. Follow this simple protocol for making a clean and cheap DIY phase separating gel.
5 Ways to Really Screw Up Your RNA Prep
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…
Cell lysis 101: 8 methods to break down cell walls
In Part 1, I introduced the types of cell walls out there and what they are made of – now it’s time to learn how to get through them. 1. Mechanical methods of cell disruption Mechanical cell disruption is really just that: forcing open the cell wall and spilling the contents. The advantage to mechanical…
Cell lysis 101: 5 types of cell walls you need to understand
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 favour of protection! Cell walls exist in…
Crush Like an Elephant, Soak Like the Rain: Old-School DNA Gel Extraction
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…
How to Calculate a DNA Primer Concentration
Calculations can be the bane of laboratory work. Fortunately, there are many easy methods to help you do the maths you need in the lab. Here, we tell you about the different ways to calculate primer concentration depending on the starting material. For all calculations, let’s assume we have 22 nmol of a DNA primer…
