Cloning Large or Complex DNA Fragments

Cloning Large or Complex DNA Fragments

Sometimes you know a project is going to be a pain before you even start it. For me, that is whenever I need to clone large (> 3 kb) or complex (e.g., a sequence with repeats) DNA fragments. Long and complex DNA fragments are more likely to create challenges during cloning. Such projects require extra care in just about…

How DNA Extraction Is Different in Plants

How DNA Extraction Is Different in Plants

I know what you are thinking, everything is made of cells, so how different can DNA extractions be in plants? The answer is… sort-of different. The overall concept is the same. Cell membranes are lysed, DNA is separated from other cell materials, washed a few times, and then resuspended in water or Tris-EDTA buffer (aka…

Viral Vector Production: Myths & Misconceptions

Viral Vector Production: Myths & Misconceptions

Viral vector production is a worthwhile skill that can be made even easier with a few tips and tricks. In general, transfection of multiple plasmids into a producer cell line results in infectious, non-replicative virus. However, it is important to ensure that your vector preparation is efficient, giving your experiments the best chance of success….

AAV Production Part II: Virus Purification

AAV Production Part II: Virus Purification

In Part I of AAV Production, I described how to produce crude (non-purified) AAV. In this article, I am going to tell you how to purify that crude prep. Virus purification is usually done by gradient ultracentrifugation. Two common methods involve gradients made from increasing concentrations of cesium chloride or iodixanol. A cesium chloride prep…

Acid Phenol Chloroform Extraction of DNA, RNA and protein:  3 in 1

Acid Phenol Chloroform Extraction of DNA, RNA and protein: 3 in 1

In austerity times, nothing is in excess. Apart from saving reagents, which can be refilled with extra financial injections, there is a commodity that cannot be easily resupplied – tissue samples! If, like me, you have experienced the fear of not having enough sample for performing a qPCR, western blot, and conventional PCR from the…

Pulsed Field Gel Electrophoresis:  Tips and Tricks

Pulsed Field Gel Electrophoresis: Tips and Tricks

Running a pulsed field gel can be exciting. It isn’t often that you get to visualize such large pieces of DNA. However, it can also be extremely frustrating. It isn’t uncommon to wait 24 hours only to find out that your DNA was degraded before you started the run. Then, you have to start all…

7 Tips for using Magnetic Beads for DNA Cleanup

7 Tips for using Magnetic Beads for DNA Cleanup

Whatever molecular biology techniques you use, at some point you will have to clean up your DNA samples to remove things like buffers, contaminants and nucleotides from you precious sample, so that you have perfectly pure DNA for your downstream experiments. Magnetic beads are one DNA cleanup option. They are simple and effective—and their reassuringly…

5 Things You Should Know About Agrobacterium Binary Vectors

5 Things You Should Know About Agrobacterium Binary Vectors

You can create stably transformed plants expressing your gene of interest; be it for the subcellular localization of your protein or simply for the in planta protein expression and purification. Whatever it is, you can do wonders with plant transformation. Sound difficult? It isn’t. Just like there are millions of microbes that interact with us,…

Cut My Gene into Pieces– Introduction to Restriction Enzyme Cloning

Cut My Gene into Pieces– Introduction to Restriction Enzyme Cloning

At the heart of cloning are restriction enzymes. Restriction enzymes are a common tool in any molecular biology lab. Need to know how large your plasmid is? Cut it with a restriction enzyme. Need to chop your genomic DNA into smaller pieces for a southern hybridization or to prepare a library? Use a restriction enzyme….

Pulsed Field Gel Electrophoresis – The Basics

Pulsed Field Gel Electrophoresis – The Basics

You have probably run a standard agarose gel hundreds of times. They are great for visualizing small DNA fragments up to 10 kb, but what if you want to examine really large pieces of DNA or even whole chromosomes? This is where pulsed field gel electrophoresis (PFGE) comes in! While the equipment required to run…

How to Plan a Restriction Cloning Experiment In Silico

How to Plan a Restriction Cloning Experiment In Silico

Restriction cloning, at its core, is quite simple. You simply cut the target vector and insert with the same enzymes, clean digested vector and insert up, ligate the two together, transform the ligated vector and insert into bacteria, and then screen. While getting each of the steps correct can be a bit of a hassle,…

Choosing the Right E. coli Strain for Transformation

Choosing the Right E. coli Strain for Transformation

Cloning, purifying, and expressing modified genetic material is routinely done in microbes such as Escherichia coli (E.coli). Relatives of this molecular biology workhorse normally live in the intestinal track of humans. The particular E. coli strain (K-12) that scientists use all over the world was isolated from the feces of a diphtheria patient in 1922.1…

SAGE Part 2: LongSAGE, RL-SAGE and SuperSAGE

SAGE Part 2: LongSAGE, RL-SAGE and SuperSAGE

SAGE, or serial analysis of gene expression, is a technique that enables you to digitally analyze the entire gene expression profile of a cell(s). Before this technique, scientists were limited to studying a few gene’s expression at once by a technique called the expressed sequence tag approach. The coolest part of SAGE is you don’t…

Vital for Soup, Vital for Labs: Serial Analysis of Gene Expression (SAGE), part 1

Vital for Soup, Vital for Labs: Serial Analysis of Gene Expression (SAGE), part 1

Some techniques can sound very dry but this isn’t one of them! SAGE was first described and published by Velculescu et al. in 1995. At the time, techniques like RNA blotting and expressed sequence tagging were used to study gene expression. However techniques like these were slow and very limited. The speed of SAGE and…

How to Choose Your Method for DNA Extraction from Whole Blood

How to Choose Your Method for DNA Extraction from Whole Blood

Over the last few decades, PCR, next generation sequencing and microarray technologies have taken blood-based research to a new level. Modern blood-based application range from DNA fingerprinting, whole genome sequencing, blood banking to liquid biopsy and many more. Regardless of the application, pure, intact, double stranded and highly concentrated DNA extraction from whole blood is…

Eight Top Tips to Maximize Yield from Whole Blood DNA Isolation
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Eight Top Tips to Maximize Yield from Whole Blood DNA Isolation

When you perform genomic DNA extraction from whole blood, low yield or low quality DNA can result in many issues. No matter your intended downstream application—qPCR, next generation sequencing, Sanger sequencing, and so on—you need high quality DNA. We’ve made this step-by-step guide to assist you in getting the highest possible DNA yield and quality, and…

Polysome Profiles:  What You Need to Know

Polysome Profiles: What You Need to Know

Has your supervisor asked you to do a polysome profile? Are you attempting them but running in to problems? This article will help explain what they are, the basics of the protocol and provide some helpful tips (including some things that I wish I had known when I first started out). Polysome Profiling: The Basics…

E.coli Electroporation vs Chemical Transformation

E.coli Electroporation vs Chemical Transformation

This is the first in a three-part series on the transformation of E.coli. By the end of this, you should be an expert on E.coli transformation and on which strains to choose for different applications. If you’re already an expert, I hope it’ll be an enjoyable refresher for you. In either case, please comment below…

A Guide to Solid Phase Reversible Immobilization

A Guide to Solid Phase Reversible Immobilization

  Scientists today depend heavily on many molecular biology techniques to perform their research. For example, with the advent of next generation sequencing (NGS): scientists are able to look at very minute details, right down to individual genetic sequence variations. However, the increase in experimental complexity means that every extra step becomes more crucial than…

Comparing Viral Vector Expression Systems

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…

How to Light Up your Life – Tips and Tricks to Troubleshoot your Luciferase Assay.
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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…

Quick reference: Determining DNA Concentration & Purity
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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…

Defeat RNAse Contamination Using Bleach in Your RNA Agarose Gel
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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…

What’s The Problem With Ampicillin Selection?
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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…

The BOOM Method for Nucleic Acid Purification: The Ultimate Chick Flick?

The BOOM Method for Nucleic Acid Purification: The Ultimate Chick Flick?

The Boom method, or Boom nucleic acid extraction method, is a solid phase extraction technique for isolating nucleic acids from a solution of biological matter. This is just a fancy way of saying you use this technique to expose and remove the nucleic acids from a cell. First developed by William R. Boom, the Boom…

There’s No Need To Be Paranoid About RNA Purification
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There’s No Need To Be Paranoid About RNA Purification

RNA purification may be a common procedure in molecular biology but it is by far the one that people fear most. Why? Dreaded RNase. It’s everywhere… all over your bench and pipettes, and floating in the air, waiting for the chance to creep into your prep, shred your RNA into nucleotides, and ruin a day’s…

5 Tips on Vector Preparation for Gene Cloning

5 Tips on Vector Preparation for Gene Cloning

One of the most crucial steps in any cloning procedure is the preparation of the vector. Get it wrong and your chances of success will be drastically reduced. The overall aim for a good vector preparation is to obtain a fairly concentrated stock of undamaged, fully digested plasmid DNA that is free from contaminants. Missing…

Get Your Clone 90% Of The Time with Ligation Independent Cloning

Get Your Clone 90% Of The Time with Ligation Independent Cloning

Are you stuck in cloning hell?, Tired of doing ligations that don’t work? Want a faster, more efficient cloning procedure? You should try ligation independent cloning. A growing number of researchers swear by ligation independent cloning methods because they are simpler and more efficient than conventional cloning and as a recent convert to their ranks,…

Southern (blot) exposure remains a useful technique
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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
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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™
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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?
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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…