How is Lab Grade Water Purified? |
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Nick Oswald |
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There’s something in the water, and it would love to go after your experiments.
Straight out of the tap, water contains microorganisms, endotoxins, DNase and RNase, salts and other impurities that could gobble up your experiment in one bite. Of course we avoid this drama completely by using purified water from which these nasties have been removed. But how is this purification done?
Well in practice a number of techniques are used, each of which can remove a different set of impurities.
So here are the techniques:
1. Distillation. A technology as old as the hills (or at least as the stills that were hidden in the hills). Water is heated to its boiling point then condensed back to liquid. This will remove many impurities but impurities with a boiling point equal to or less than that of water will also be carried over in to the distillate.
2. Microfiltration. In this technique, pressure is used to force the water through a filter with pore sizes of 1 to 0.1 micron in order to remove particulate matter. Filter diameters lower than 0.2 micron removes bacteria – so-called cold sterilisation.
3. Ultrafiltration uses even smaller pore sizes (down to 0.003 micron). These are essentially molecular sieves, which remove molecules with a diameter larger than the pore size. It can be used to remove viruses, endotoxins, RNase and DNase
4. Reverse osmosis. If you thought that ultrafiltration used impressively small pore sizes, you’ll be even more impressed by reverse osmosis . Reverse osmosis filters have pore sizes of less than 0.001 microns, which allows them to sieve ions depending on their diameter. This is used for desalting the water.
5. Filtration through a bed of activated carbon is useful for removing things like chloride ions and organic compounds, which are adsorbed onto the surface of the carbon.
6. UV radiation. We all know what UV radiation, at specific wavelengths, can do to DNA and microorganisms. So UV is an obvious way to remove microorganisms from the water. It can also clean up the water by breaking down certain organic compounds into less harmful products.
7. Deionization/ Ion exchange. This technique removes ions from the water by passing it through a resin bed containing a mixture of cationic and anionic resins. Positive ions in the water are attracted to the anionic resin particles and negative ions are (yes, you’ve guessed it) attracted to the cationic resins. The result is that nicely deionised water comes out of the other end of the resin bed.
Commercially available water, or water purification systems will typically use a combination of these. The higher the water purity grade, the more techniques used.
Any questions or comments? Just jump in and join the discussion in the comments section below. The water’s lovely.
The Basics: How Ethanol Precipitation of DNA and RNA Works
How SDS-PAGE works
5 Laboratory Sterilisation Methods
The Best of Bitesize Bio 2009 |
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Nick Oswald |
To enable tagging you will need to register on Bitesize Bio. We're sorry for the inconvenience, but it's free, only takes a few seconds, and it will enable you to view our seminars for free, ask questions from the professional community, and take part in the lively community of Bitesize Bio
Before we get our brains fully back into gear after the festive season and embark on what we hope will be a momentous year for Bitesize Bio (watch this space, we’ve got some cool stuff in store), we thought this would be a good time to look back at the best articles we published in 2009.
Below are the 10 most popular articles that were published in the last year. Enjoy.
1. Free Online Bioinformatics Tools – Carrie topped the chart with a great article describing 4 must-use, free bioinformatics resources.
2. How to Create a Heatmap in Excel – A neat Excel hack from Paul, showing how to bring your numerical results to life in glorious technicolor.
3. Quantitative RT-PCR: One-step or Two-step RT? – Shoba weighed in with some excellent practical advice for quantitative PCR
4. An Intro to Cell-free Protein synthesis – And Shoba wasn’t finished there — she also produced this excellent beginners guide to cell free protein synthesis.
5. Reasons to be a Scientist Part II – If you want to reassure yourself that science is a great career choice, have a look at this one.
6. Delivering Effective Criticism – This is a message for anyone who is or will be a supervisor in any capacity. Think about how you treat your charges!
7. Plasmid v Genomic DNA Extraction:The Difference – Another molecular biology mystery demystified by Suzanne
8. 5 Types of Bad Boss and How to Handle Them – And some great practical advice from Suzanne on how to deal with your bad boss.
9. How To Get Great DNA Sequencing Results – Because bad sequencing results are such a waste of time.
10. 10 Tips for Consistent Real-Time PCR - And not content with only two entries in the top 10, Shoba sneaks in a 3rd with this excellent tip sheet.
I hope that these will help your brain wake up for the new year. If you have any personal favorites, please share them in the comments.
As I said, look out for some more great stuff from us in the coming year. And if you want to be a part of it and join our writing team, the door (or, rather, the contact form) is always open.
Best of luck with your research in 2010.
Get Involved With Bitesize Bio
Assessing the Bitesize Bio Experience
The essential conferences of 2009
How To Make Fewer Mistakes In The Lab |
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Nick Oswald |
To enable tagging you will need to register on Bitesize Bio. We're sorry for the inconvenience, but it's free, only takes a few seconds, and it will enable you to view our seminars for free, ask questions from the professional community, and take part in the lively community of Bitesize Bio
How often do you make errors in the lab that ruin a good experiment? Rather than flaws in experimental design, I mean errors like forgetting to add a reagent, pipetting the wrong amount or following a protocol step wrongly.
Especially early on in your career, errors like this can be a real drain on your productivity. As I talked about earlier, listening to music in the lab may or may not help you here, but here are 10 other ways in which you can reduce your error rate and get more results.
1. Use a checklist. I have always found that using checklists during experiments is a great way to focus the mind and stop me from forgetting to add something or doing things in the wrong order. But you don’t have to just believe me… a recent study in the New England Journal of Medicine showed that the use of simple checklists during surgery cut deaths and complications by one third. So perhaps by adopting checklists you can reduce the attrition rate for your experiments.
2. New protocols and SOPs: write out your own version. When you are performing a new protocol, the worst thing you can do is just jump in and get started without gaining a knowledge of the whole procedure first. That is just asking for things to go wrong. So one rule I always keep is to write out my own version of the protocol in Word before I start, using a standard format. This gives two benefits. Firstly, it forces you to read through the whole protocol and secondly it gives you a standardised copy of the protocol that you can keep, annotate and make checklists from.
3. Annotate. If you make a mistake in a protocol, annotate your copy so that you won’t make the same mistake again.
4. Repetitive pipetting: be consistant and use bookmarks. If you are doing a large experiment with a lot of repetition, just letting your mind wander for a few seconds can ruin your experiment if you don’t put in safeguards. One example is where you have a whole rack of tubes into which you are repetitively pipetting — it’s so easy to get lost and forget where you were. You can guard against this by being rigidly consistant with your pipetting order and by bookmarking your progress by closing the lid (or similar) after you have pipetted.
5. Don’t multitask too much. Multitasking is essential in the lab, but should be practiced with care as overdoing it can make you error-prone. More on this here.
6. Get set up before you start. This is obvious, but it’s often neglected… Make sure you have everything you need before you start. The moment you wander off to pick something up you are inserting an error-inviting distraction into your procedure.
7. Prepare in bulk. For experiments you perform often, it pays to put in a bit of time upfront to prepare for a lot of future experiments in advance. Make stock solutions in bulk (aliquoted if required), prefill tubes with reagents so you can just take them out of the fridge/freezer and go, and create experiment forms to save you from writing out the same stuff in your lab-book over and over again (for more on this click here). The more you can prepare upfront, the less room there is for error later.
8. Don’t spend so long in the lab. Ok so you need to put in the hours to make sure you generate results but there is a tradeoff because spending too long in the lab will dull your sharpness. Where the cut-off lies is a personal thing.
9. Get enough sleep. Just like too many hours in the lab will dull you, so will lack of hours in bed. Get some sleep!
10. Take responsibility. Especially if you are an early career scientist, problems can occur if you expect others to be responsible for your work, for ensuring that you have everything you need etc. Taking the viewpoint early on that the buck starts and stops with you will empower you to make sure that you create the situation where your experiments have the optimal chance of working.
Those are my thoughts on reducing your error rate in the lab. What approaches do you use?
Error Bars in Biology
Don't Overdo The Multi-tasking
How Accurate are Your Pipettes?
Which is Best: TAE, TBE or Something Else? |
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Nick Oswald |
To enable tagging you will need to register on Bitesize Bio. We're sorry for the inconvenience, but it's free, only takes a few seconds, and it will enable you to view our seminars for free, ask questions from the professional community, and take part in the lively community of Bitesize Bio
TAE or TBE, which is best? Well, of course, it depends on what you want to do. Here are the pros and cons of both:
But you might be better of using neither of these buffers. Despite the fact that they have been firmly established as the most popular buffers for DNA electrophoresis since their emergence in the early 1970s, TBE and TBE are not really optimised for the job and have a lot of disadvantages.
Borate and acetete are simply carry-overs from earlier work on protein electrophoresis buffers upon which the technology of DNA electrophoresis was built. And while the reasons that Tris was chosen as the primary cation by early pioneers of DNA electrophoresis are not clear, the draw-backs certainly are. As well as being expensive, Tris-based buffers are not ideal for DNA electrophoresis because, even under optimal conditions, they are prone to a temperature-current feedback loop that causes the solution, and therefore your gel, to overheat. And finally, EDTA is a relic from RNA electrophoresis, which has been unecessarily retained in DNA electrophoresis buffers.
In an earlier article here on Bitesize Bio, Bala described new and improved DNA electrophoresis buffers developed by scientists at John’s Hopkins University. These are optimised for the job and are faster, cheaper and less prone to overheating than TAE and TBE.
For more information on this, check out this article, which details the history of DNA electrophoresis buffers and the development of the new buffers. The authors of this paper (and the inventors of the new buffers) have started a company called Faster, Better Media to manufacture and distribute the buffers.
So what do you normally use; TBE, TAE, or something else?
Faster, Cooler DNA gels
Sharpen Up Your Protein Gels
A Menagerie of Mini Prep Methods
Does (Should) Your Lab Rock? |
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Nick Oswald |
To enable tagging you will need to register on Bitesize Bio. We're sorry for the inconvenience, but it's free, only takes a few seconds, and it will enable you to view our seminars for free, ask questions from the professional community, and take part in the lively community of Bitesize Bio
My PhD was a soul-less affair. It was also rock-less, jazz-less and pop-less. And all because my supervisor was of the opinion that music in the lab was a distraction that reduced concentration and our ability to do the job. “Rubbish!”, I thought, “Nothing helps you through a mindless task like splitting cells, pipetting or labelling like a bit of music”.
A 2005 study in the “Psychology of Music” Journal backs up my opinion, reporting that music increased the performance of software developers in both creative and routine tasks. But over the years I have found that while music in the lab does help alleviate the boredom in some situations, and even inspire you in others, it can also cause serious headaches.
Hell is other people’s music
Passive soundwaves can seriously damage mental health. What if your lab mates’ idea of wonderful music is the Titanic theme, Enya or Alanis Morissette (you know, the one who has no idea what ironic means)?
While they are working and warbling away to their favorite tunes, you could be trapped day in day out in a Music Hell that certainly won’t improve your productivity or your mood. And sometimes you just need silence in which to do your work – should you be forced to auditorily inhale if others want to partake? Probably not.
So it’s vital that your lab agrees on an audio policy that includes whether to allow music in the lab. when it is allowed and what type, or how to agree on it. At the top of the policy should be a cast-iron rule that if any one person in the lab doesn’t want the music to be on, then the sounds should be firmly switched off.
What to play
When the music is on, there is the small but thorny matter of what to play. The common but, to me, extremely depressing, solution is to go for a generic radio station. This is favored because these types of radio station cater for the lowest common denominator; they play music that not too many really people hate. But the huge downside this is the sort of music that no-one really likes either. It’s difficult to be inspired by bland pop, over-played oldies and a playlist that numbers in 10′s rather than 100′s. In my opinion, you’d be better off with no music at all.
But it doesn’t have to be that way. Ideas like theme days, where everyone brings in a CD from their collection to match a given theme, or nominated DJs, where each person gets to plug their iPod into the speakers and take control of the music for a certain time, can be a lot of fun and create a sense of community in the lab. Or using services like LastFM or Spotify you can create customised, and even trained, playlists to suit the collective tastes of your lab. It doesn’t matter how you choose your lab music. It just has to be democratic and fair.
What about headphones?
Another possible solution is to use headphones to personalise and control your in-lab musical experience. This gets rid of many of the irritations with playing music in the lab, but creates its own problems because it isolates lab members from one and other, and the tinny sound of “Ironic” being played at full volume in a pair of headphones can be almost as annoying as the speaker-borne experience. So your lab’s audio policy should also deal with headphones.
So while music has its benefits in the lab, making sure that it doesn’t annoy, alienate or isolate other members of your lab takes a bit of consideration. Perhaps my PhD supervisor was right after all.
What do you think?
BioPop: 10 Songs That Should Be On Every Biologist's iPod
Church Scaremongering on Stem Cells
Stem Cell Century: The Law of a Controversial Science
A Halloween Treat: 10 Molecular Bio Tips |
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Nick Oswald |
To enable tagging you will need to register on Bitesize Bio. We're sorry for the inconvenience, but it's free, only takes a few seconds, and it will enable you to view our seminars for free, ask questions from the professional community, and take part in the lively community of Bitesize Bio
This Halloween we want to treat you with some molecular biology goodies in the shape 10 juicy tricks. We’ve been bringing you advice since August 2007, but some of our readers may just be discovering our hallowed grounds. Join with us in this bringing some of these old tricks back to life.
1. Save money by recycling electroporation cuvettes
Article: Re-cycling Electroporation Cuvettes
2. Ditch the LB and get rich cultures for super-productive plasmid preps by using PDM, a stoichiometrically optimised medium for plasmid production.
Article: Pimp Your Plasmid Growth Medium
3. Using LB-grown cultures for IPTG induced expression? Lactose in the LB will give a low level of background expression. Switch it off by adding 0.3% glucose.
Article: Shut of Background lac Promoter Expression in LB”>
4. Ever noticed that when you do the PE wash during gel extractions, a droplet of PE tends to stubbornly rest inside the column at the side that was to the outside of the centrifuge rotor? Get rid of that droplet, and stop it carrying through during elution, by turning the tube 180 degrees so the droplet is on the inside, and giving it another quick centrifugation.
Article: 5 More Tips for DNA Gel Extraction
5. Eppendorf tubes sent in standard envelopes can get stuck in mail sorting machines. So when you are sending plasmids in the mail either use a padded envelope, which doesn’t go through the sorting machine, or soak the plasmid in a small piece of filter paper, wrap it in cling film and send it flat packed!
Article: Sending Plasmids: How to Avoid Jail Time and Shredded Envelopes
6. Did you get too many bands in your PCR reaction? Here is a fast and easy method to rescue your PCR. Run the PCR reaction on a gel to separate the products and then stab the band of interest with a 20G syringe needle a few times. Swirl into a new PCR reaction and 20 cycles later, you’ve got your single band ready for cloning or sequencing. This is fast, easy, and much less expensive than PCR optimization.
Article: PCR Rescue: Making One Band From Many
7. Another good way to eliminate non-specific PCR amplification is the Touch-Down PCR (TD-PCR) technique. TD-PCR allows for amplification of only the amplicons with the highest primer-template complementarity.
Article: Touchdown PCR: a primer and some tips
8. Want to become the lab MacGyver? Here is a top ten list of lab MacGyverisms; ways to use everyday items to make gadgets and low-tech solutions for the lab. Examples include using McDonald’s straws (they are autoclavable!) for disposable pipettes and cheap liquid handsoap for blot washes. Setting up a lab at home seems very feasible after reading this article!
Article: Low Tech Lab Gadgets: My All Time Favs
9. There are numerous competent E.coli strains to choose from. For clarity on the benefits of all the different mutations and then how to choose the right strain, this article outlines the options simply and clearly.
Article: Choosing a Competent E.coli Strain
10. TBE sucks. Run gels for 10 minutes at 200 volts using sodium boric acid buffer instead. 10 mM Sodium Boric acid stays cool even at high voltage, allowing gel gels to run 7 times faster!
Article: Faster, Cooler, DNA Gels
Have any more tricks of your own? Share them with us in the comments, or using the contact form. If you’ve got a good one we’ll write about it — or you are always welcome to contribute an article yourself.
Happy Halloween!
E.coli Electroporation vs Chemical Transformation
The Best of: Tech Tips
5 More Tips for DNA Gel Extraction
Don’t Miss This Free Online Bioconference |
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Nick Oswald |
To enable tagging you will need to register on Bitesize Bio. We're sorry for the inconvenience, but it's free, only takes a few seconds, and it will enable you to view our seminars for free, ask questions from the professional community, and take part in the lively community of Bitesize Bio
How would you like to participate in a free, online Bioconference? Watch and participate in live presentations from the biggest names in the life science industry and the thought leaders of the research community? And do it all from the comfort of your own computer?
Well Bioconference Live allows you to do just that on 17-19 November (2009). And I think this is a not-to-be missed event. As well as lining up some great speakers, the Bioconference Live team have worked hard to capture the conference experience, offering the opportunity to field questions to speakers, view posters, mingle in the networking lounge, and even visit virtual vendor booths (we’ll have a booth there so be sure to come and chat to us!).
Topic tracks include Bioinformatics, Microbiology, Nucleic Acid Analysis and Amplification, Drug Discovery and Delivery, Lab Automation, Cell & Tissue Analysis, Biotechnology, Autism Research, Cancer Research, Immunology, Clinical & Diagnostic Applications, Chemistry, Neuroscience, Protein Expression, Genetics and Genomics. With all of these choices, you’re bound to find alot of interesting stuff, no matter what your research interests are.
Enter the lobby
Your conference experience begins when you log in and enter the lobby of the virtual convention center. This is a 3D envirornment from where you can feel the conference vibe and access all of the available options, just like in a traditional conference, although you’ll have to supply your own coffee and cookies.
Speeches, talks and posters
The lobby houses aisles containing hundreds virtual booths, each of which houses live keynote speeches or talks/posters that are going on continuously for the three day event. Enter any of the booths at the advertised time to watch a live talk, then hang around afterwards for moderated text chat with the speaker. Immediately after all talks and presentations are given live, they’re then available on-demand (for free) to come back and see later, so no need to worry if you miss something.
Network
Networking is a vital aspect of any conference, so Bioconference Live has a Networking Lounge where users can congregate to meet with their peers or network with other professionals in their field, then use the Live Chat feature to discuss industry issues and news, or just have a conversation.
Visit the exhibit hall
You can then visit the Exhibit Hall and explore new technologies. Companies are divided by specialty or floors, so you are able to see all the vendors within your area(s) of interest in one central location. Each vendor has their own “virtual booth”, where you can view their commercials or video uploads, peruse their pdf’s (adding them to your “briefcase” if it is something that you would like to refer back to later or download to your PC), and even Live Chat with a representative from their company if you have any questions or would like to request additional information. Suzanne and myself will be at the Bitesize Bio booth and we’d love to see you there.
Register now, win a prize
You can register for Bioconference Live by clicking here. Once registered, you can enter a prize draw (with prizes including a Macbook) by telling three friends about the conference. I’m really looking forward to this event, and hope to meet you there.
Inspirational Talks at TED
Free Online Bioinformatics Tools
Don't Forget to Visit the Exhibits!
Go Huddle Around The Culture Dish |
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Nick Oswald |
To enable tagging you will need to register on Bitesize Bio. We're sorry for the inconvenience, but it's free, only takes a few seconds, and it will enable you to view our seminars for free, ask questions from the professional community, and take part in the lively community of Bitesize Bio
Spearheaded by Bitesize Bio Superstar Suzanne Kennedy, our good friends at Mo Bio Laboratories have started an excellent blog called The Culture Dish, which is well worth taking a look at.
MoBio’s expertise is in developing technologies for nucleic acid isolation and purification from environmental microbial samples, so their blog focuses on that domain. It may be only 5-articles-old but there is some compelling stuff in there already, including:
The also have a wonderful section called “Where in the World?” that visits some of the scientists who use Mo Bio’s products all over the globe.
Company blogs tend to be a bit dry, but Suzanne and the Mo Bio team manage to keep it light, fun and (dare I say?) Bitesize Bio-like, making it a pleasure to read.
So go and huddle around The Culture Dish with Suzanne and Mo Bio and let us know what you think of it.
Even Better Free Molecular Biology Software: Serial Cloner |
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Nick Oswald |
To enable tagging you will need to register on Bitesize Bio. We're sorry for the inconvenience, but it's free, only takes a few seconds, and it will enable you to view our seminars for free, ask questions from the professional community, and take part in the lively community of Bitesize Bio
In 2007, I wrote a couple of articles on the best free software for molecular biologists on Mac and PC. One of the highlights of those lists was a fantastic cross-platform for in silico DNA cloning, sequence analysis and visualization, called Serial Cloner (move over VectorNTI!).
The creator of Serial Cloner, Franck Perez, contacted me recently to tell me that he had just released an excellent new version of the software and asked if I would relay this to our readers. Well, as a big fan of Serial Cloner I am only too happy to oblige.
If you do molecular biology and you haven’t tried our Serial Cloner, I’d urge you to give it a go. It is very intuitive and is packed with features; from basics like constructing importing sequences, constructing plasmid maps and restriction mapping, through more complex things like sequence alignment, Gateway cloning and siRNA design.
Among the many improvements Franck has made in this new version are:
Check it out at http://serialbasics.free.fr/Serial_Cloner.html
10 links: Free Mac Software for Molecular Biologists
10 links: Free PC Software for Molecular Biologists
Free, Publication Quality Plasmid Annotation
Spare a Thought for the Old Scientists |
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Nick Oswald |
To enable tagging you will need to register on Bitesize Bio. We're sorry for the inconvenience, but it's free, only takes a few seconds, and it will enable you to view our seminars for free, ask questions from the professional community, and take part in the lively community of Bitesize Bio
You youngsters don’t know how easy you’ve got it. Kits, outsourcing and improved practices are making research easier and easier. At least in theory (who are we kidding?).
In the old days things were much tougher, and many wiley old scientists bear the scars, mental and physical, of carrying out techniques that were mind numbing, frustrating or just plain dangerous. Now the Bitesize Bio team is (generally) not THAT old – or so we tell ourselves – but we put our heads together and to think of the techniques we are most glad we don’t have to use anymore. And, together with a some suggestions from our Twitter followers, we came up with a top 10 .
Please feel free to share your own, especially if you are a REALLY old scientist and remember some of the especially nasty stuff!
1. DNA sequencing. In the old days DNA sequencing meant pouring massive gels, slopping around up to your armpits in P-32 phosphorus and reading the sequence by hand from the gel. We much prefer the “stick a tube in an envelope and send it off to the sequencing service” approach.
2. Making Oligos. Another revolution. Sending the sequence off to the oligo synthesis service is so much better than spending an eternity to (attempt to) make them yourself.
3. Phenol-chloroform extractions. No more smelly phenol in our labs, we’ve got spin columns (Thanks @Kyrsten_Jensen for this suggestion)
4. Packing protein purification columns. Buying them pre-made is so much easier (Thanks @Kyrsten_Jensen for this suggestion)
5. Electrophoresis with home-made electrophoresis apparatus. Electrifying! (although our home-made gel visualisation apparatus is pretty good!)
6. Northen Blots. Thank you bioinformatics.
7. Drying Agarose gels/SDS-PAGE gels for archiving. Thank you digital photography! (thanks @saxphile for this suggestion)
8. Adding mineral oil to PCR. Whoever invented heated lids in PCR machines was a genius (thanks @saxphile for this suggestion)
9. Home made minipreps. I may not have been the greenest-fingered scientist that ever lived but the failure rate of my home-made minipreps was pretty big, especially when I did many of them at one. Thank goodness for miniprep kits.
10. Pouring your own SDS-PAGE gels. This one, and I suppose many of the others above that advocate buying pre-made stuff, is controversial but in my experience is very cost-effective to buy in pre-made SDS-PAGE gels rather than pour your own. No wasted time on leaking gel apparatus or forgetting to add the Temed and the gels are nice and uniform.
So that’s we don’t miss doing in the lab. How about you?
Spare a Thought for Tech Support Reps
A Classification System For Scientists
Sharpen Up Your Protein Gels
