I often wonder why it is that molecular biology researchers stubbornly refuse to change 40 year old methods that, while they work, are not as good as newer, faster and cheaper methods out there.

I suppose rational scientists often have irrational superstitions.

One example of an old method that could be improved is the growth media used for plasmid preparation.

The majority of us, throughout our university careers, have used either SOC, LB or TB, for recombinant plasmid propagation, typically in E. coli. LB or Luria-Bertani broth has been in use for almost 60 years or thereabouts, while SOC has certainly been in use for 2 decades.

But by adding in a few more ingredients or being more economical on others (especially yeast extract and tryptone) that you could get a higher plasmid yield, quicker and with less money. Read more »

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- hydrolysable analogue of lactose, the natural inducer of the lac operon.

Tight control of expression from the lac promoter, which is required if the protein being expressed is toxic to the E.coli host or for a variety of other reasons, is not possible when using LB because it contains lactose.

But how does lactose get into LB? Read more »

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 up to 20 times… perhaps more a guesstimate than a real number, but hey, who’s complaining?

Read more »

Today I was browsing through the “new technologies” section on the Biocompare website. Apart from the amazing but super-expensive automation equipment that most of us unfortunately have little chance of getting our hands on (at least at the moment), five products caught my eye as being useful for improving techniques widely used by researchers. I hope you find some, or all, of them of interest to you… Read more »

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 gels to a maximum of 5-10 volts/cm (e.g 100 volts for a 10 cm gel) and extend the running time, sometimes to hours.

Although long gel runs, like long restriction digests, are often used as a convenient coffee break opportunity they can also eat into the molecular biologist’s precious time, leading to longer and less efficient working days.

But, in 2004, a team of scientists from Johns Hopkins came up with solutions (pardon the pun) to this problem. They have developed and verified three conductive buffers that stay cool during electrophoresis, allowing the voltage to be racked up to a massive 35 volts/cm without any problem, reducing the time taken to run gels by up to 7 times. Read more »

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 for better DNA Gel extraction proved very popular. It seems like many of us are keen to get all the tips we can on this procedure. Well, if it’s tips you are looking for, we are always happy to oblige.

By scouring the recesses of my brain, colleagues and the internet I have squeezed out 5 more tips on DNA gel extraction. Maybe one of them will make the difference for you. Read more »

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.

However, there is a quick & dirty screening method that is even faster than colony PCR.

Called colony cracking, it involves picking a colony straight into lysis buffer, running the extract straight onto an agarose gel, and then identifying positive clones based the electrophoretic mobility difference between the super coiled DNA with and without insert - plasmids with inserts will travel slower than plasmids without them (no-insert control).

Obviously, the larger the insert, the easier it will be to distinguish between positive and negative clones, but researchers claim to be able to detect inserts of only 200bp (see the alternate protocol links at the bottom of the page).

If you’d like to try this method, read further. Read more »

For routine procedures involving cell lysis, it’s good for the lysis to be… routine. Of course there are many good and freely available lysis buffer recipes but for convenience and reproducibility you can’t beat pre-made lysis buffers.

Focusing on lysis for protein extraction, here are some of the reagents available for fast and efficient lysis of some of the most common cell types you might be using. Read more »

A while back, I wrote an article on 5 DNA ligation tips that could improve the efficiency of your cloning procedures. It proved to be quite a popular article so here are another 3 tips that might make your ligations even better! Read more »

Last week, in my article about the perils of exposing DNA to UV light during cloning procedures, I mentioned a couple of stains that offer an alternative to ethidium bromide for DNA visualisation.

I this article I compare all of the available DNA stains (I know of) that can be used in electrophoresis to clarify the options available to you. Read more »