Owner: Bitesize Bio. The Molecular Biology Blog URL:http://www.bitesizebio.com Join Date: Tue, 28 Aug 2007 17:43:53 -0500 Rating:0 Site Description: Technical tips, updates and comment for molecular biologist. The tricks for getting the best out of PCR, plasmid cloning, oligos, ligation. Site statistics:Click here
My favorite PCR polymerase 2007-08-28 17:00:44 What do you get when you cross a Pyrococcus DNA polymerase with a dsDNA binding domain? …a highly processive, high fidelity, lighting fast PCR work-horse
Those Finnish wizards at Finnzymes have made that work-horse a reality with their Phusion polymerase. I had been meaning to try out this enzyme for quite a while, but never got around to it, then a problematic 11 kb amplification brought it back to mind. (more…)
Cut & Paste with Quikchange 2007-08-28 07:11:54 I recently had a problem where I needed to cut out an expression cassette (a promoter coupled to a coding sequence) from one vector and paste it into another a vector containing an expression cassette so I could get tandem expression of the two genes. The recipient vector had no suitable restriction sites for performing this operation so I was faced with the fairly lengthly task of introducing two new restriction sites on the recipient vector using quikchange, then amplifying the expression cassette and sub-cloning into the recipient. Not much fun. (more…)
Zyppy Plasmid Miniprep Kit 2007-08-24 02:49:02 Zymo research have launched a new plasmid miniprep kit that not only is has the coolest name of any kit on the market but is, the manufacturers claim, the fastest means of plasmid purification available. (more…)
RNA Polymerase II Assayed in Living Cells 2007-08-24 02:37:47 A Nature Structural & Molecular Biology article published by Singer et al has provided a fascinating insight into the kinetics of RNA polymerase II during transcription. (more…)
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Quick reference: Determining DNA concentration & Purity 2007-08-22 06:14:09 The most comprehensive way to evaluate DNA concentration and purity is to use both UV spectrophotometeric measurements and agarose gel eletrophoresis. This quick reference
guide gives an overview of the information that can be derived from both. (more…)
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5 Tips on Vector Preparation for Cloning 2007-08-22 05:31:30 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 any of these aims will affect the efficiency of the ligation and is the cause of many of the problems researchers have with cloning. Here are my top tips for vector preparation: (more…)
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, Cloning
Codon Usage Prediction 2007-08-31 10:19:28 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 in the organism will reflect this codon usage bias. (more…)
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, Prediction
Better Pubmed Journal Searches 2007-08-31 04:57:04 Ever get too many hits from your Pubmed searches? Using field tags allows you to generate more specific searches than keywords alone, saving you from trawling through hundreds of irrelevant articles. (more…)
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DNA replication, transcription and translation in action 2007-08-29 16:34:38 While browsing you tube I came across this really nice animation of DNA replication, transcription and translation complete with imaginative sound effects. I hope you enjoy it as much as I did. (more…)
Low cost DNA gel documentation 2007-08-29 03:24:09 Equipment for photographing DNA gels stained with ethidium bromide (or other fluorescent dyes), doesn’t have to cost thousands of dollars. These days, great pictures can be obtained with a standard digital camera with an orange filter. Here’s how. (more…)
Plasmid Cut & Paste with Quikchange 2007-08-28 07:11:54 I recently had a problem where I needed to cut out an expression cassette (a promoter coupled to a coding sequence) from one plasmid vector and paste it into another containing an expression cassette so I could get tandem expression of the two genes. The recipient vector had no suitable restriction sites for performing this operation so I was faced with the fairly lengthly task of introducing two new restriction sites on the recipient vector using quikchange, then amplifying the expression cassette and sub-cloning into the recipient. Not much fun. (more…)
The World’s Fastest Miniprep? 2007-09-20 10:18:15 I love this great time-lapse video entitled “a day in the lab”. If only minipreps were this fast and easy! (more…)
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Competent E.coli: To buy or not to buy? 2007-09-20 09:39:46 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 can be prepared easily in the lab (for protocols see here and here). (more…)
15 Reasons to Be A Scientist 2007-09-18 06:56:20 Just for fun, here my top 15 reasons for being a scientist. Add your own reasons in the comments below if you so wish.
Not being stuck behind a desk all day every day
Conferences… see the world for free (more…)
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E.coli Electroporation vs Chemical Transformation 2007-09-17 19:01:46 This is the first in a three part series on the transformation of E.coli. By the end of this you should be an expert on E.coli transformation and on which strains to choose for different applications. If you’re already an expert I hope it’ll be an enjoyable refresher for you. In either case, please comment below if you have anything to add. (more…)
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One Tube PCR Cloning Method 2007-09-14 05:27:58 I love shortcuts, and this one is very good. Chun-Ming Liu of Plant Research International has a number of molecular biology protocols on his website, but my favourite is his One Tube PCR Cloning
Method. The protocol involves simply putting the vector, insert, restriction enzyme, ligase (and in his case polymerase for polishing the ends) into one tube, incubating at 16°C for 2 days and then transforming. (more…)
Binder CO2 incubator - movie fun 2007-09-14 03:46:16 Binder have released a fun movie
entitled “Germfellas” to promote their latest CO2 incubator, which comes with inbuilt 180°C sterilization capability. The movie is nicely done and is a great piece of marketing. Take a look if you have the time.
10 Tips for Better Presentations 2007-09-12 18:02:03 I have been at a conference today and don’t have too much time to write this, so this will be a quick article. After watching lots of speakers of varying competence I though that it would be good to outline some tips for great presentations. Speaking is an integral part of a scientist’s job, and honing these skills will is great for both your career and your confidence. Here is my 10 cents worth - feel free to add more of your own tips in the comments field below: (more…)
What’s The Problem With Ampicillin Selection? 2007-09-11 07:24:44 Ampicillin is commonly used as a selection marker for plasmids in gene cloning and protein expression in E.coli and other bacteria. While it serves it’s purpose, there can be problems using this selection marker if the user is unaware of it’s limitations. This article provides a quick overview of what these limitations are and how to avoid them. (more…)
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The Dark Side of Gene Synthesis 2007-09-10 08:41:37 After writing my recent article on custom gene synthesis, I came across this article in the excellent Seven Stones systems biology blog that highlights the potential dark side of this emerging technology. The article describes a recent Nature Biotechnology commentary by Bügl et al 2007 in which executives from the DNA synthesis industry discuss regulation of their industry in order to address biosecurity concerns. (more…)
10 links: Free PC Software for Molecular Biologists 2007-09-10 05:02:10 Here is a list of 10 pieces of great molecular biology software for PC users that I hope you will find useful. I am not a regular PC user myself so I have not tested all of these out so if any of these are no good, or if you have any favorites you’d like to add, please let me know or leave a comment. If you are a Mac user, click here for the Mac list. (more…)
Read more:links
, Software
, Molecular
10 Do’s and Don’ts for PhD Students 2007-09-27 10:21:31 My PhD is rapidly becoming a distant memory. Before nostalgia completely obscures my recollections of this chapter of my career, I thought I’d jot down some pointers for prospective and current PhD students. These are mainly based on things I wish I had done during my PhD, or mistakes I have seen others make. I hope they’ll make your life easier! If you have any other suggestions, please feel free to chip in with a comment.
(more…)
You Know You’ve Been In the Lab Too Long When… 2007-09-26 08:18:19 This has been doing the rounds all over the web, so I thought I’d post my 10 favorites.
You know you’ve worked in the lab too long when…
You wash your hands before you go to the toilet
You tell your family to store the milk “at 4°C” (more…)
Ethidium Bromide: A Reality Check 2007-09-26 06:15:38 The hysteria among molecular biologists about our old friend ethidium bromide has long been an irritation to me. Researchers are rightly wary of this potential carcinogen. More recently this wariness has been whipped up into a witch hunt by companies touting “safer” alternatives and disposal methods. While I don’t for a minute think that we should all throw our gloves away and bathe in the stuff, I think that it’s time for an informed reality check about the dangers, and the myths about ethidium bromide. (more…)
Read more:Check
, Reality Check
Protein Expression with a Cherry on Top 2007-09-24 06:32:43 If you do a lot of heterologous protein expression, take a look at Eurogentec’s Cherry
Express Kit. Based on the T7 expression system, the CherryExpress vector has a sequence encoding a small red polypeptide (the heme binding part of cytochrome) fused to the promoter. When your favorite gene is cloned in, the resulting fusion protein is bright red, allowing easy identification of clones expressing your gene. Even better, protein expression level can be quantified at 480nm and the highly soluble peptide could even improve the soluble expression of your protein. Tasty.
Choosing a Competent E.coli Strain 2007-09-24 05:52:45 Of all the of competent E. coli cell strains available, which one should you choose? The choice of strain to use in a given experiment is determined in large part by the nature of the experiment and the set of traits that best fit it. In this article I summarize some of the most important traits and their benefits in downstream applications. (more…)
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Plasmid archiving at Addgene 2007-10-04 06:25:49 Addgene is a non-profit plasmid repository that stores and distributes plasmids for academic labs. It’s great if you work in an academic lab and they happen to have your plasmid - drop them an order and get your plasmid in the mail. I don’t work in an academic lab, but I still love addgene… here’s why… (more…)
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An Attractive Genomic DNA Isolation Kit 2007-10-03 09:22:20 I was becoming a bit bored with the tedium of column-based kits, so when I had to isolate genomic DNA from a range of micro-organisms for a recent project I decided to try something new. Invitrogen’s ChargeSwitch genomic DNA isolation kit, which uses magnetic beads to separate the gDNA from the cell debris, seemed interesting so I thought I’d give it a go. (more…)
Read more:Isolation
Antibiotics Used in Molecular Biology 2007-10-02 05:43:28 Antibiotics are used in a wide range of techniques in molecular biology. My aim with this post is to provide an easy reference to some of the main antibiotics used in molecular biology, their mechanisms, range and working concentrations. I hope you will find it useful. If I have missed out an antibiotic that you use routinely in your work, please feel free to contact me or leave a comment and and I will add it to the table. (more…)
Read more:Antibiotics
, Molecular
, Molecular Biology
Why Do Enzymes Have Optimal Temperatures? 2007-10-11 08:44:33 Every biologist is familiar with the profile of the rate of an enzymatic reaction versus temperature as shown in the figure. We know that enzymes from E.coli or warm-blooded animals tend to have an optimum around 37°C while those from thermal vent bacteria have much higher optimal temperatures. Surprisingly, I find that many biologists don’t have a grasp of why enzymes have these temperature profiles. Actually it’s reassuringly simple. (more…)
Read more:Enzymes
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