How to Prevent False Results in Colony PCR
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How 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…

CPEC– a Quick and Inexpensive Cloning Strategy

CPEC– 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…

Cloning Methods: 5 Different Ways to Assemble

Cloning 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…

Bacterial Transformation Troubleshooting for Beginners

Bacterial 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…

Oligo Purification Methods: How, Why and for What?

Oligo 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…

Get Your Polymerase Cycling Assembly Oligos Together

Get 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…

Outsourcing Research:  Should Your Experiment Spend Some Time Away from You?

Outsourcing 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…

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….

Three Important Things to Check After Obtaining Your Plasmid

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…

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,…

Get Ready, Get Set, Retro – How to Get Started With Retroviral Transduction

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 Shut Off Background Lac Expression in LB

How 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….

Six Facts About Restriction Enzymes

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…

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…

Long-Range PCR:  It’s About Choosing the Right Enzyme

Long-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…

Seamless Ligation Cloning Extract (SLiCE) Explored and Explained

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…

Polymerase Incomplete Primer Extension (PIPE) Cloning Method

Polymerase 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…

Am I Damaging My  E. coli by Spinning at High Speeds?

Am 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…

Banish the Background with Toxin–antitoxin Cloning Systems

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…

10 ways to improve blunt-end ligations

10 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…