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 »

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 »

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 »

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 »

This is a story that could strike fear into your heart if you use UV light to visualize DNA that you later intend to clone. Read on if you dare.

A while back I was doing a project where I had to make a mutation library of a plasmid. There are a number of ways to do this but I thought I would be smart. UV light is mutagenic - why not just put the plasmid on a transilluminator for a while? That should introduce some mutations and make my library with the minimum of fuss.

Well it was a reasonable idea, but the results were a real eye-opener. Read more »

Yawn….

The awards season is upon us once again. Overpaid, under-worked and over-ego’d celebrities get together to slap each other’s backs and tell each other how great they are.

But little do they know where the real party in town is. The 2008 Thermostable Polymerase Awards (the THEPA’s) are underway and you have a front row seat.

Here are the winners: Read more »

After years of loyalty, our relationship was becoming stale - things just weren’t the way they used to be. I was putting in more than I was getting back and complaining about it didn’t seem to help. I just got the same old answer thrown back at me… “it’s not our problem, it’s yours”.

Then things changed. I don’t know how it happened, it just did. We met on the internet and I immediately knew this could be the answer to my prayers. I said I just wanted my routine DNA-handling procedures to be uncomplicated, they said “the beauty of science is to make things simple”.

With shaking hands I pressed the e-mail “send” button - I had just ordered free samples of Zymo Research’s Zyppy plasmid miniprep, DNA clean-up and gel extraction kits. It felt so good. Would this be the start of something new and wonderful? The end of low quality DNA samples? The end of poor yields? I hoped so. Read more »

Lysis of some microbes is easy, but for others its much more difficult - I think due to differences in the make-up of their cell wall.

At the moment, one of my colleagues is preparing hundreds of cell-free extracts from microbes isolated from the environment to screen for interesting enzyme activities. Since she is processing a wide range of microbes she has to use a method that will lyse all of them - even the most difficult ones.

Unfortunately that means using glass beads and a lot of vortexing to mechanically break the cells. I say unfortunately because not only does it take her a very long time to process all of her samples, but she has a very noisy vortex mixer, which is driving me crazy!

Yesterday I came across something that could be the answer to both of our prayers. Read more »

I just came across a neat device now being offered by BioRad that may interest those of you who do a lot of electroporation of difficult-to-transfect mammalian cells, where tedious optimization of the electroporation protocol itself is required. Read more »

One of the most common tasks in molecular biology is cleaning up DNA from aqueous solutions to remove buffer salts, enzymes or other substances that could affect downstream applications. Examples include cleaning up PCR reactions, digests or other enzymatic treatments and cleaning up genomic or plasmid DNA contaminated with cellular proteins/debris. There are several ways to approach DNA clean-up, here are five of them. Read more »