Posts Tagged ‘Cloning’
The Beginner’s Guide to Reading Plasmid Maps
Plasmid maps are a cornerstone of biology, but they are confusing to read for beginners. Our easy guide tells you how to read them, where to look for essential information, and how to avoid common mistakes.
Read MoreOverhang PCR: Add Missing DNA Sequences Using Primers
Need to add extra nucleotides to your plasmid or other DNA sample? Here’s how to use overhang PCR to easily add extra bases using primers.
Read MoreA Beginner’s Guide to How Blunt-End Cloning Works
Struggling with your cloning? It could be time to try blunt-end cloning. This easy introduction explains what it is, when to use it, and how.
Read MoreUnderstanding The Optimum Temperature For Enzymes
Do you fully understand why enzymes have the best catalytic activity within a specific temperature ranges? Find out in our handy guide.
Read MoreMultiple Fragment Ligation: The Why and How
You may be familiar with standard single fragment ligations, but did you know you can ligate multiple fragments into your vector all at the same time! Discover how to perform multiple fragment ligation, including the different methods and troubleshooting tips for when things go wrong.
Read MoreHow (and Why) To Manipulate Plasmid Copy Number
Here we take a closer look at plasmid copy number and examine how it can be manipulated in the lab giving you flexibility in your work.
Read MoreDNA Ligation: How it Works & 6 Top Tips
Understanding the basic (and simple!) chemistry behind DNA ligation will help you get better DNA ligation results. Learn all about it here.
Read MoreHow to Make Your Own Chemically Competent Cells
We’ll show you how to make a DIY stock of chemically competent E. coli, the workhorse in the molecular biology laboratory.
Read MoreEssential Factors for Successful Gateway Cloning
Want to use Gateway cloning or having trouble using this technology? Find out how it works and get helpful tips to increase your success.
Read MoreHow to Prevent False Results in Colony PCR
How to Prevent False Results in Colony PCR Colony PCR saves time and reduces costs by eliminating the need for plasmid purification. However, confounding results abound — but only if you fail to anticipate them. This article outlines the major perpetrators of false results and how to prevent them. For a more general overview of…
Read MoreCPEC– a Quick and Inexpensive Cloning Strategy
Cloning Strategies – a Whole Lot of Options to Choose Molecular cloning has come a long way from simple restriction digestion-ligation cloning strategies to a large number of highly efficient alternatives. Broadly classified, cloning techniques can be divided as sequence dependent and sequence independent strategies. Sequence-dependent strategies are based on restriction digestion-ligation techniques or site-specific…
Read MoreCloning Methods: 5 Different Ways to Assemble
Over the past few decades molecular biologists have developed procedures to simplify and standardize cloning processes, allowing vast arrays of artificial DNA structures to be more easily assembled. Are you familiar with all the cloning options out there? Let’s look at five different cloning methods you can use to get your construct. At the end…
Read MoreBacterial Transformation Troubleshooting for Beginners
The first time I did a transformation was when I worked with site directed mutagenesis. I cloned a protein sequence into the p15TVL vector, created my mutants (but that’s another story), and was finally ready for the next step: transformation and expression of my desired protein. Little did I know that my enthusiasm would fall…
Read MoreCodon Optimization 101
The intriguing thing about protein expression is that the combination of transfer RNAs (tRNAs) that translate the 3 letter codon into an amino acid (aa) far exceeds the number of existing amino acids (aa). If you do the math correctly, the maximum number of unique combinations using the triplet code to code for the 4…
Read MoreOligo Purification Methods: How, Why and for What?
Who amongst us hasn’t had the need for oligonucleotides in an experiment? It is a cornerstone in many procedures and techniques. Depending on the goal, it can be very hard to design just the right oligo for your experiment. Oligos must have the right length; the right amount of C-G, T-A; they can’t form secondary…
Read MoreGet Your Polymerase Cycling Assembly Oligos Together
The polymerase chain reaction (PCR) is the backbone of many lab techniques. In short, it allows for the exponential amplification of a specific segment of DNA. Through the use of primers encoding restriction enzyme sites, these amplified fragments are used in downstream cloning procedures, usually leading to the insertion of one, maybe two, PCR fragments…
Read MoreOutsourcing Research: Should Your Experiment Spend Some Time Away from You?
As a researcher, it’s satisfying to manage your own projects and do the bench work yourself. After all, if you don’t have experience with a technique, you’re usually expected to figure it out (with or without direct supervision). In some situations, dealing with difficult molecular techniques is simply part of the job description. The scientific…
Read MoreCut 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.…
Read MoreDemystifying REBASE and NEBcutter
Two years ago, all I knew was third BASE in my baseball field and the cutter ball from the pitcher. Now, I know a lot more about lab-based BASES and cutters: REBASE and NEBcutter. While they sound like baseball terms, REBASE and NEBcutter are tools for working with restriction enzymes. Read on to find out…
Read MoreGet 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,…
Read MoreGet 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…
Read MoreHow 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…
Read MoreHow to Shut Off Background Lac Expression in LB
Here’s a tip that you may find useful if you are expressing proteins in E.coli using a lac promoter-based expression system, e.g. pET, in LB medium (L-broth). Lac expression systems are typically induced in the lab using IPTG (isopropyl-beta-D-thiogalacto- pyranoside), which is a non- hydrolyzable analog of lactose, the natural inducer of the lac operon.…
Read MoreSix 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…
Read MoreHow 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.
Read More5 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…
Read MoreLong-Range PCR: It’s About Choosing the Right Enzyme
The ability for DNA polymerase to copy a long stretch of DNA is becoming increasingly important. Why? It has to do with the advances in our sequencing technologies. Our next generation sequencing (NGS) technology requires the DNA polymerase to copy a long stretch of DNA (sometimes up to 50kb) as NGS is churning out genetic…
Read MoreCRISPR 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.
Read MoreThree 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…
Read MoreA Molecular Biology Roadmap For First Time Travelers
The more experienced hands in your lab know that molecular biology is rarely just a journey from A to B. As a result, I’ve constructed this short workflow as an introduction to genomic molecular techniques.
Read MoreGet To Know Your DNA Polymerases
While most may think standard Taq is the backbone of PCR, many other DNA polymerase options exist. The polymerase you use significantly impacts the efficacy of your PCR, specifically on the product yield, the purity of the product, and the faithfulness with which the starting product is transcribed. Sometimes, these matter less, and quick and…
Read MoreSeamless 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…
Read MorePolymerase Incomplete Primer Extension (PIPE) Cloning Method
PIPE PCR is a ligase-independent, restriction enzyme-free cloning strategy like SLIC (link to my SLIC article), SLiCE and CPEC. The PIPE method eliminates sequence constraints and reduces cloning and site mutagenesis to a single PCR step followed by product treatment. It is fast, cost-effective and highly efficient. The key step is designing the primers; one…
Read MoreAm I Damaging My E. coli by Spinning at High Speeds?
Dear Aunt Yersinia, A very annoying postdoc in our group keeps telling me off for spinning E.coli at 13K in a tabletop centrifuge. The postdoc claims that high speed damages cytoskeleton and this will reduce my transformation frequency. But I don’t believe her as the cells are cushioned by water during centrifugation. Can you tell…
Read MoreCodon Usage Analysis
Problems with expressing your gene? One of the potential stumbling blocks in heterologous gene expression is incompatible codon usage. Every amino acid can be encoded by more than one codon, and for every amino acid each organism has a favorite codon that it tends to use more often than the others. The availability of tRNA…
Read MoreBanish 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…
Read More10 ways to improve blunt-end ligations
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…
Read MoreSequence and Ligation Independent Cloning
Could ligation independent cloning get any better? Well here is a technique that is sequence AND ligation independent.
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