Tech Clinic #4: Can a single E.coli take up 2 plasmids?

Tech Clinic #4: Can a single E.coli take up 2 plasmids?

The following question was emailed to Bitesize Bio by Beheroze Sattha and I gladly took up the challenge, and I immediately knew the answer. Or so I thought. After delving extensively into Pubmed, Genes V (I know, I need a new version) and Molecular Cloning I have come up with an answer, but it is…

Tech Clinic #3: DNA digestion, precipitation and clean-up

Thanks to Bitesize Bio reader, Muthu Arumugam for contacting us about some problems he has been having with restriction digestion and clean up of DNA. I have boiled his query down to four main questions that are pertinent for most molecular biologists, so I hope that Muthu and everyone else can learn something from my…

Tech Clinic #2: Gel Extraction – Avoid/Rescue a Bad 260/230 Ratio

Tech Clinic #2: Gel Extraction – Avoid/Rescue a Bad 260/230 Ratio

Gel extraction — what could be easier? Now we have quick and easy gel extraction kits, we no longer need to use time-consuming old fashioned methods like electro-elution or “freeze and squeeze”. Thank goodness. When Gel Extraction Goes Wrong But even the simplest of procedures can go wrong. Maybe you were distracted, confused, or thought…

Controls and Tips for TA cloning

Controls and Tips for TA cloning

Controls are obviously extremely important when setting up experiments. Without them, meaningful interpretation of the experimental results can be impossible. I say obviously, but in my previous job as a technical services scientist, you’d be surprised at how often I found myself talking to customers about the importance of controls. One customer commented, during our…

Six Important Factors for Successful Reverse Transcription
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Six Important Factors for Successful Reverse Transcription

The reverse transcription (RT) step of RT-PCR for converting RNA to cDNA is critical for accuracy in quantification and for finding low copy messages. Thus, you want to make sure that this step is performed with the highest efficiency but without having to optimize every single step. To help you further in optimizing the RT…

A Quik Way Around Partial Restriction Digests

A Quik Way Around Partial Restriction Digests

No matter how many times you look at it, it’s not going to change. You are planning your next cloning experiment, but there’s a problem. The only restriction enzyme that cuts in a suitable position on your plasmid vector also, as luck would have it, cuts in another position elsewhere in the vector so you…

The Best Way to Desalt DNA for Electroporation

After ligation, the method you use for desalting your sample prior to electroporation is critical, especially if your ligation is inefficient, according to a study by Schlaak et al [1]. Under standard electroporation conditions, the electric field of 12-18 kV/cm generated in a 0.1mm-gap electroporation cuvette means that the conductivity of the sample must be…

How To Get Great DNA Sequencing Results

There is nothing more frustrating than getting back rubbish data from a DNA sequencing run, especially when you are waiting for an important result. For example, confirmation of that clone you have been trying to get for the past three months! A lot of the time, the quality of sequencing data is within your control….

Recycle Those DNA Extraction Columns

Recycle Those DNA Extraction Columns

You know those ridiculously priced and throw-away DNA mini, midi and maxi-prep columns? Well the good news is that you can actually re-use them if you are reasonably careful at regenerating them, with this simple and cheap method described in detail by Nagadenahalli B. Siddappa in Biotechniques in 2007. Apparently these columns can be reused…

Faster, Cooler DNA gels

All over the world, molecular biologists are tragically wasting hours of their life running DNA gels using tris-based conduction buffers like TBE or TAE. These buffers are known to overheat at high voltages, causing problems with gel integrity, sample denaturation and more. Because of this, molecular biologists are forced to keep the voltage of their…

5 More Tips for DNA Gel Extraction

Problems with DNA gel extraction can be a real show-stopper since this is such a routinely used procedure. But, even if you are having no particular problems, it’s always nice to try and pick up some information that might improve your technique just that little bit. Probably for these very reasons, Suzanne’s article 10 Tips…

Quick and Dirty Screening for Cloned Inserts

Quick and Dirty Screening for Cloned Inserts

For identifying positive clones from a plasmid cloning procedure, the routine of performing a mini-prep and then checking the putative clones by restriction digestion is most commonly used. Of course, if you need to screen a large number of clones, another option is a colony PCR to identify positives, followed by restriction digests to confirm….

Ethidium Bromide: The Alternatives
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Ethidium Bromide: The Alternatives

How can you avoid the perils of exposing DNA to UV light during cloning procedure? Use an alternative DNA stain! Ethidium bromide is not your only option. In this article, we will compare the available DNA stains that can be used in electrophoresis to clarify the options available to you. Ethidium Bromide The classic DNA…

Sending Plasmids: How to Avoid Jail Time and Shredded Envelopes

Sending Plasmids: How to Avoid Jail Time and Shredded Envelopes

Whether you need to get your plasmid DNA to a lab on the other side of the world, or a few hundred miles down the road, it’s important to make sure your precious sample gets there, it is not degraded, and you don’t end up in jail. Here’s the Bitesize guide on how to send…

History of Molecular Biology

History of Molecular Biology

As a freshman biology major in undergrad, I was introduced to molecular biology with the following description: Molecular biology represents the intersection of genetics, biochemistry and cell biology. Some people, it turns out, add microbiology and virology into the mix. So molecular biology is often used as a catch-all, to describe a wide breadth of…

Solved: Heterologous Gene Expression Problems

Solved: Heterologous Gene Expression Problems

When heterologous gene expression goes wrong it can be a real headache. Here’s my checklist for the steps to take when you encounter problems with this dark art. 1. Check the construct by sequencing the expression cassette to make sure that everything is as you expect. A lack of expression could result from a stray…

Competent E.coli: To buy or not to buy?

Competent E.coli: To buy or not to buy?

Buying competent cells from commercial suppliers is convenient, provides a guarantee of quality, and gives access to strains with a variety of in-built traits that assist with things like maintenance of plasmid integrity (more on these traits later). However, this can be an expensive business. Alternatively, competent cells of any strain, including the specially-constructed commercial…

Easier Gene Cloning With Positive Selection Cloning Vectors

Easier Gene Cloning With Positive Selection Cloning Vectors

Isn’t it a pain digesting, purifying and dephosphorylating your cloning vector prep to eliminate prevent high background in your ligation/transformation? A new generation of positive selection cloning vectors promises to eliminate all of that hassle by killing off any vector that has not taken up the insert you are trying to clone. Positive selection cloning…

Custom Gene Synthesis: A PCR alternative.

Artificial gene synthesis was first reported in 1972 when a group of researchers at Massachusetts Institute of Technology synthesized a complete yeast alanine tRNA gene. Synthesis of the first peptide- and protein-encoding genes ensued in the following decade. Since then, synthetic biology has advanced in leaps and bounds, and custom gene synthesis, a one-time expensive option for…