dnature Blog

Come in spinner

Sun, 14 Apr 2013 20:11:32 +1200

That last little job before running your qPCR - no, not trying to remember which random sample you loaded in the wells; rather, centrifuging down the reactions to the bottom of the wells. This removes air bubbles and ensures all reaction components are combined.

You can use 96 well plates centrifuges (expensive) or now there are purpose built smaller models as well - still hitting close to $1,000. Therefore, welcome to what we call in our lab the System Amplification Loading Aid Device (SALAD).

That's right, a salad spinner. Eco qPCR users will know it well but
we've used it for 96 well and 384 well plates with very good results.

Look at the features! :

- Fast

- spin multiple plates

- Reaches ??? g in 5 seconds.

- Rapid braking (stop cranking it!)

Some will lash a plate holder to the side of the bowl to hold the plate but with many 96 well plates and the Eco plates, we can wedge them easily into the slots. I prefer the ones with handles rather than the pull-strings . . .only because I used to keep breaking the string and having to replace it !

Look out for the dnature version coming soon - with the word 'scientific' on it somewhere, it should be under $300

The best microbiology

Thu, 10 Jan 2013 09:35:13 +1300

Welcome to 2013 - I hope it's a great year for you.
Toward the end of last year, we were pleased to sponsor the NZ Microbiology conference in Dunedin. We also added some personal sponsorship in the form of a tour to (and through) Emerson's brewery. After doing something similar many years ago (same conference, same brewery!), a number of the people had said they had enjoyed that particular part of the conference. It was interesting to note that some of those same people joined this tour as well.

At the risk of waxing nostalgia, I have had a long affiliation to the brewery - my tastebuds about 5 minutes longer than that. As a member of the Biochemistry department at University of Otago, we were accorded some special treatment in the early days of the brewery. Indeed, Richard Emerson had brought his first Emerson's t-shirt to show on this recent tour.

Richard Emerson holding the first Emerson's t-shirt from 1993:

"We drank the first barrell"

I still have my version - the one I like to think must be rarer, in black - as a momento of these early days.

It's amazing to see how the brewery has grown from those early days but one thing has always remained constant - the gumboot-clad, nose-in-the-hops, beer-chewing master brewer that is Richard.

A(nother) fascinating tour with a liberal tasting at the end meant some happy conference delegats at the end of the tour. I gave Richard a dnature t-shirt at the end, in recognition of the early t-shirt I had received from him. Perhaps selfishly? Richard gave me an early t-shirt and his company has grown in leaps and bound (and in sweat and toil, granted). So if I gave Richard a t-shirt then . . .

Presenting Richard with a dnature t-shirt

Thanks to the Emerson's crew and Jane and Richard of course, for the opportunity for NZ microbiologists to see the best of applied microbiology. Thanks also to Anne Dahse (NIWA) for the photos above and thanks to everyone who came along - we hope you enjoyed it.

More photos

Richard explaining the process

tasting time !

tasting aftermath - microbiologists are thirsty people

two better than one

Mon, 10 Sep 2012 10:25:01 +1200

Working with RNA viruses (and multiple isolate strains), sometimes it can be hard enough to get sufficiently conserved sequence for primers (and harder if designing a TaqMan or probe-based assay). However a recent example in our lab showed the benefit of designing multiple assays for the same target and comparing the performance across template dilutions.

We were asked to look at virus X in some samples (name changed - unless you're a potato - which actually has a virus X. But this wasn't it). No published qPCR assays for this virus so I designed 2 SYBR-based assays on an alignment of sequences. Pretty standard stuff. Both amplicon sequences were checked for secondary structure before the primers were ordered from Biosearch.

Short story from there:

RNA extracted from samples (and positive control material)
cDNA made (qPCR-perfect Quanta cDNA supermix then diluted 1:10)
SYBR FastMix qPCR across dilutions - both assays using 0.3uM primers and a 2-step protocol.

Results?

One set of primers (1732) detected the virus at a much earlier (lower) Cq than the other set (1310) - indicating better analytical sensitivity (although the absolute sensitivity was similar).

Amplification of viral cDNA dilutions with two pairs of primers (1310 and 1732)

The melting peaks showed the presence of a lower Tm 'shoulder' - likely a region of DNA breathing or melting prior to the rest of the amplicon.

Comparison of melting peak data with two pairs of primers (1310 and 1732)

Optimisation of annealing temperature may have improved this (PCR additives didn't) but having the second assay (one specific peak) meant the major assay optimisation for 1310 primers consisted of ditching and forgetting them.

Another example perhaps of 'how do you know whether you have the best assay if you only have one?'.
Thoughts?

Is that the economics of science? Or vice versa

Thu, 07 Jun 2012 20:58:07 +1200

As part of the Transit of Venus forum in Gisborne and a Radio New Zealand series, I was lucky enough to attend today's panel discussion on whether science is the answer to greater economic prosperity. Actually there was little luck involved to attend - I live in Gisborne, the panel discussion was free and the venue was 3 minutes from the lab with parking over the road. Yes, the advantages of living in Gisborne!

The panel was led by Kim Hill and included Prof Peter Gluckman (science advisor to the PM), Derek Handley (who sold mobile advertising company The Hyperfactory to large USA media company), Professor Shaun Hendy from the MacDiarmid Institute and economist Dr Caroline Saunders from Lincoln University.

A number of ideas were discussed - DH:that 80% of science should be focussed on particular problems (cf 'blue sky' research); PG: NZ needed to diversify away from commodity based exporting and more value-added - and gave examples where that was happening (Tatua and lactoferrin).
The main proposal put forward - science and R&D needed a major 'step-change', rather than incremental improvement (which, compared with the rest of the world does little to stop us sliding further behind). "What if we fail?" As the rest of the world betters its own efforts, by doing nothing (or doing little) we are already failing and slipping further behind.

The gap - the wide chasm - between businesses and scientists/researchers was raised. Are scientists in NZ generally seen as white-coated, antisocial 'boffins' who shouldn't be let out of their laboratory? In the early 90s, scientists were the first to adopt email. I recall an article on science that mentioned 'they (scientists) were keen to know if you had an email address yet'. The popular reason given was that scientists would rather email than stand up and talk to their colleagues.

Having being involved with the unfortunate kiwifruit Psa issue I can say there were several positive things from it. One of those positive things was the opportunity to work with some other open, collaborative scientists and researchers - as well as businesses who appreciated science and the efforts involved.

The forum discussion was lively (thanks to the deliberate provocations of Kim Hill), entertaining and provoked a lot of thought. it demonstrated that the potential to shake up science, move it closer to businesses (where needed) and make invaluable leaps may not be far away.

How our products come about

Sat, 14 Apr 2012 09:45:13 +1200

Many of the products/equipment we distribute have come about from personal experience. We either tried them, liked them . .and found no NZ distributor; or else we've bought the equipment for our own lab (and subsequently become distributors). But sometimes it comes about from a customer gripe. She'd taken time from her busy manager duties to gown up, glove up to demonstrate cutting some bands out a gel - only to find all the scalpel baldes have disappeared.
That reminded me of a product I'd seen (imagined?) and while looking for it - came across a simple tool for rapid band excision from gels. The X-tracta is the latest addition to the dnature range. . . .but within our walls anyway, we'll be calling them Lisas.

2012 - and 20 years of PCR

Tue, 24 Jan 2012 19:13:13 +1300

Happy 2012 to everyone. Of course I relate most things to PCR stuff (well, this is a PCR blog) and this year marks 20 years since I first did a reaction. Which means it's 20 years since my first blank gel and 20 years since I amplified strange bands, wrong-sized bands - before hitting the conditions and getting it right.
Or at least for the positive control. . .

Things I remember?

While developing a new method, being given a tube of Taq, buffer and MgCl2 and following a paper (it used the same single primer)
PCRs then were in 0.5ml tubes and about 100µl volumes
Since I was only running 10µl on a gel, I figured I didn't need the other 80µl immediately chopped all volumes 5-fold (including the enzyme) to 20µl reactions
2 drops of oil as the overlay (yep, no heated lid). Plus greasing the tubes with the same oil on tissue paper before seating over the machine with the car headlamp as the heating element.
Another student speeding up the tube greasing by dropping oil straight into the wells. Since the wells weren't solid, much smoke resulting as the oil heated on the lamp!
Back then Taq was about $1.20 a unit. Now we're under 20 cents a unit (check out FIREPol)
No hotstart enzymes. Hotstart - when I used it - was adding the MgCl2 to the reaction tube during a pause in the first annealing step.
My positive control was just under 3kb. That was considered a long amplicon and I was asked from time to time how I got my 'big PCR' to work by others (ironically, low enzyme amounts would later be shown to be useful for longer PCRs). But then we were amazed to read of enzyme blends going out to 35kb.

I consider myself lucky to learn about PCR then and learn some of its fun attributes. Thanks Iain.
(I would later take some small perverse delight in sending papers to my former supervisor that made the method I developed obsolete)

You still remember your own first PCR reactions? Please share your memories. . .

Drumroll please. . .

Sun, 02 Oct 2011 22:19:10 +1300

The envelope please - this year's IgNobel awards have just been announced. The research that makes you laugh - then makes you think. The winner for 2011 in no particular order:

PHYSIOLOGY PRIZE: A multinational study "No Evidence of Contagious Yawning in the Red-Footed Tortoise."

Ref: 'No Evidence Of Contagious Yawning in the Red-Footed Tortoise Geochelone carbonaria," Anna Wilkinson, Natalie Sebanz, Isabella Mandl, Ludwig Huber, Current Zoology, vol. 57, no. 4, 2011. pp. 477-84.

CHEMISTRY PRIZE: the ideal density of airborne wasabi (pungent horseradish) to awaken sleeping people in case of a fire or other emergency, and for applying this knowledge to invent the wasabi alarm.
REFERENCE: US patent application 2010/0308995 A1. Filing date: Feb 5, 2009.

MEDICINE PRIZE: that people make better decisions about some kinds of things — but worse decisions about other kinds of things‚ when they have a strong urge to urinate.

Ref: "Inhibitory Spillover: Increased Urination Urgency Facilitates Impulse Control in Unrelated Domains," Mirjam A. Tuk, Debra Trampe and Luk Warlop, Psychological Science, vol. 22, no. 5, May 2011, pp. 627-633.

Ref: "The Effect of Acute Increase in Urge to Void on Cognitive Function in Healthy Adults," Matthew S. Lewis, Peter J. Snyder, Robert H. Pietrzak, David Darby, Robert A. Feldman, Paul T. Maruff, Neurology and Urodynamics, vol. 30, no. 1, January 2011, pp. 183-7.

PSYCHOLOGY PRIZE: Karl Halvor Teigen of the University of Oslo, Norway, for trying to understand why, in everyday life, people sigh.

Ref: "Is a Sigh 'Just a Sigh'? Sighs as Emotional Signals and Responses to a Difficult Task," Karl Halvor Teigen, Scandinavian Journal of Psychology, vol. 49, no. 1, 2008, pp. 49–57.

LITERATURE PRIZE: John Perry of Stanford University, USA, for his Theory of Structured Procrastination, which says: To be a high achiever, always work on something important, using it as a way to avoid doing something that's even more important.

Ref: "How to Procrastinate and Still Get Things Done," John Perry, Chronicle of Higher Education, February 23, 1996. Later republished elsewhere under the title "Structured Procrastination."

BIOLOGY PRIZE: The discoverythat a certain kind of beetle mates with a certain kind of Australian beer bottle

Ref: "Beetles on the Bottle: Male Buprestids Mistake Stubbies for Females (Coleoptera)," D.T. Gwynne, and D.C.F. Rentz, Journal of the Australian Entomological Society, vol. 22, , no. 1, 1983, pp. 79-80

Ref: "Beetles on the Bottle," D.T. Gwynne and D.C.F. Rentz, Antenna: Proceedings (A) of the Royal Entomological Society London, vol. 8, no. 3, 1984, pp. 116-7.

PHYSICS PRIZE: For determining why discus throwers become dizzy, and why hammer throwers don't.

Ref: "Dizziness in Discus Throwers is Related to Motion Sickness Generated While Spinning," Philippe Perrin, Cyril Perrot, Dominique Deviterne, Bruno Ragaru and Herman Kingma, Acta Oto-laryngologica, vol. 120, no. 3, March 2000, pp. 390–5.

MATHEMATICS PRIZE: Dorothy Martin of the USA (who predicted the world would end in 1954), Pat Robertson of the USA (who predicted the world would end in 1982), Elizabeth Clare Prophet of the USA (who predicted the world would end in 1990), Lee Jang Rim of KOREA (who predicted the world would end in 1992), Credonia Mwerinde of UGANDA (who predicted the world would end in 1999), and Harold Camping of the USA (who predicted the world would end on September 6, 1994 and later predicted that the world will end on October 21, 2011), for teaching the world to be careful when making mathematical assumptions and calculations.

PEACE PRIZE: Arturas Zuokas, the mayor of Vilnius, Lithuania, for demonstrating that the problem of illegally parked luxury cars can be solved by running them over with an armored tank.
(yep, video reference on YouTube)

PUBLIC SAFETY PRIZE: The series of safety experiments in which a person drives an automobile on a major highway while a visor repeatedly flaps down over his face, blinding him.

REFERENCE: "The Attentional Demand of Automobile Driving," John W. Senders, et al., Highway Research Record, vol. 195, 1967, pp. 15-33. VIDEO

Thanks to www.improbable.com - and leave a comment on your favourite award. The mayor and his tank has the visuals but sometimes you just have to hand it to the Aussies and their beer habits!

New Zealand scientist "on a par with Galileo"

Mon, 01 Aug 2011 21:13:51 +1200

An interesting article on Allan Wilson (with the words used in this subject line) in the latest issue of NZ Listener - promoting the upcoming lecture tour by the PhD student who, with Wilson, published on "mitochondrial Eve" - our common female ancestor from Africa.

Allan Wilson was the first researcher outside of Cetus (the company where PCR was developed) to use PCR. Yep, back in the time when it was 3 water baths and repeated enzyme addition. Or as they wrote in 1987:

. . .in a total volume of 100µl. The mixture was heated for 10 min at 950 C to separate the strands, spun for 10 sec in an Eppendorf model 5414 microcentrifuge to bring down condensate and cool the sample, and then cooled for an additional 2 min at room temperature. Two µl of DNA polymerase I Klenow fragment (0.5 units/µl) were added and the reaction was allowed to proceed for 2 min at room temperature. The reaction mixtures were then heated again for 2 min at 95°C and the cycle just described was repeated a total of 20 times."
Nucl. Acids. Res 15:2 529-542

Sheesh. We've got it easy now eh?

However it seems appropriate that Wilson was the first to use PCR outside the Cetus laboratories. Many of his PhD students had previously gone from his Berkeley lab to Cetus where they were heavily involved in directing the development of early PCR. One of these students - Tom White - bought a chemist friend on board Cetus to help synthesis oligonucleotides (no PCR, no "primers"!?). That chemist with the molecular biology PhD was of course Kary Mullis who would go on to conceive of PCR.

Other students of Allan Wilson include Svante Paabo (pioneer of ancient DNA analysis and now director of the Max Planck Institute for Evolutionary Anthropology - the institute where the Neanderthal genome was recently sequenced). Russell Higuchi - on the early PCR papers using Taq polymerase - would later develop real-time qPCR while at Cetus and later at Roche Molecular Systems. The original qPCR though? That was also Russ Higuchi - but in his earlier work on cloning the first sequences from an extinct animal: the quagga.

So in the large whirling atmosphere of PCR and qPCR - Allan Wilson appears at the heart of it all. I hope to get along to one of Rebecca Cann's lectures in NZ that describes his legacy - check out here for dates.

And the final link with Allan Wilson and PCR? Mullis was only able to study for a PhD thanks to the influence of his advisory committee (oh, and a Nature paper as a grad student). And on that advisory committee of 3. . . Allan Wilson.

dnature in the news

Mon, 09 May 2011 12:27:33 +1200

dnature (and John) recently appeared in a couple of press articles on the development of new tests to rapidly identify the virulent (Psa-V) from the less-virulent strain.

One article is here (Gisborne Herald) and the other is here (Bay of Plenty Times).

What sort of person. . . .?

Sat, 02 Apr 2011 11:10:48 +1300

Working on a new assay that needs the best performance possible, the question strikes me 'what sort of person goes through minor primer/probe/whatever adjustments to see curves improve by half to 1 cycle'. By that I mean you can spend considerable time doing primer chessboards (the most painful) or even probe titrations just to see slight improvements. . . or not.

In workshops, I ask participants the question 'what does it take to do PCR?' - and yes, by that I mean successfully. After pipettes and the ability to use them accurately, I reckon the ability to follow a recipe ("your cake turns out the same way each time") features highly.
But that's performing a PCR or qPCR reaction - I'm talking about the work that goes into developing new assays.

The time taken to calculate the slight reagent concentration changes, the time and effort to set up reactions with those changes, probe titrations then plus and minus tweakers. . .the list can carry on.
So what sort of person does it take to develop these new assays? Add a comment or three. . .

How stable is 'stable at room temperature'

Sun, 06 Feb 2011 12:28:15 +1300

Well, not wishing to brag as the temperature heads over 30 degrees again but the warm weather here in Gisborne made me think of the question we're often asked about the PCR reagents we sell from our European manufacturer Solis BioDyne. Yes, all their reagents (dNTPs, ladders, PCR masters etc) are stable at room temperature for up to a month and we're often asked 'how stable are they really?)
Solis regularly demonstrates the stability of their enzymes and the hot weather here made me think of a recent demonstration they showed - and kindly let me reproduce here. As they say, a picture speaks. . .

The team has a habit of travelling to various places around the world with reagents in their luggage. No packaging, no chilling - just the tubes. After trips they're then run in conventional or qPCR applications. So on a trip to Egypt. . . .

PCR reagents in foreground at Mit-Rahina outdoor museum

PCR reagents at the Sphinx

After the trips, the reagents are then assessed against reference reagents. In this case, qPCR shows the same Cq for samples with reference and 'well-travelled' reagents and thus the performance is the same, demonstrating that the 'travelled' reagent retains full activity.

qPCR results between masters

I might just have to take some reagents down to the beach with me next time. Or to this place: Anaura Bay, just north of Gisborne. Just back from a few days camping over Waitangi weekend in this spectacular area, when this photo was taken by Gisborne photographer and artist, Laura Scheper

Welcome to 2011 !

Tue, 25 Jan 2011 19:46:57 +1300

Another year is upon us and yet, writing this, we're already heading rapidly towards the end of January. Toward the end of last year the blog writing became non-existent when 3 letters entered the NZ vernacular and many people got very busy. Those 3 letters are Psa (or Pseudomonas syringae pathovar actinidiae). Unfortunately a potentially high damage pathogen was now threatening the 1.5 billion dollar industry. The rapid detection of this bacteria throughout kiwifruit regions in NZ indicated that the pathogen had been here for some time and thus it was likely that the disease had been managed inadvertently by many growers.

But there's nothing like hearing the words "we have an incursion" to spark up a Saturday afternoon. Since then it's been solid work to design and develop a new hydrolysis probe (OK, TaqMan probe) assay for this pathogen for screening purposes before duplexing it with an internal control. A great opportunity to fire up the Eco qPCR system 'in anger' as it were. Using this new instrument with the Quanta FastMix reagents we've also just launched means the assay has been running in 40 minutes - very handy when trying different parameters during the optimisation.

I hope everyone's start to 2011 is away with a hiss and a roar and I look forward to writing down notes or applications of interest. As always, feel free to add any comments on the sort of PCR/qPCR notes you'd like to see or drop us a line.

Save time! no freezing required!

Sun, 31 Oct 2010 14:07:45 +1300

No, the title is not the promotion for a product but information on one of our habits. So it's getting towards 5pm and you've added your salt of choice and ethanol/isopropanol for your nucleic acid precipitations. The freezer makes a convenient holding place for your tubes and you're off home.

But for any other time of the day, the requirement to freeze your samples for 1 hour/ 1 morning/ 1 night seems one of those tightly held dogmas of molecular biology that. . .that. . . make little difference except to our waiting time. Yep, add your salt and alcohol of choice, invert several times to get the homogeneous suspension and head for the centrifuge. The length of time spinning the samples looks to be the differentiating factor.

A great discussion on the topic here at BiteSize Bio and more information here. For a high throughput plant viroid extraction process I developed, I also found by qPCR that freezing samples made no difference to viroid recovery and extraction time was greatly reduced by going straight to centrifugation. Add a comment on your own favourite method of precipitating DNA or RNA and how long (or short) a time you freeze your samples.

making one band from many

Mon, 11 Oct 2010 09:36:00 +1300

This article was originally published at BitesizeBio here
It’s the molecular biologist’s version of ‘I have good news. . and bad news’.
The good news is that I amplified the DNA band of interest. The bad news is that I amplified these other bands as well! Oh, and this smear. What to do?
Typically you might try and cut out the band of interest and gel-purify – an issue if it’s a lower intensity product and most of the DNA is lost in the subsequent purification. Or the products are more closely spaced than your dexterity allows.

An alternative I’ve used for many years is band-stab PCR. This is an excellent technique to specifically re-amplify the DNA of interest and increase yield. With only one product generated in the subsequent PCR, the remaining PCR reaction volume can be rapidly cleaned up without gel purification.

How to do it?

1. When I notice multiple bands, I put my gel aside and go and set up a new reaction (I make up a 50µl reaction if I want a lot of this fragment). I add water in place of the usual DNA volume.

2. I then go back and place my gel on the transilluminator and gently blot off moisture with Whatman 3MM paper or Kimwipes.

3. Using either lower power or longer wavelength U.V. to avoid DNA damage, I then take a 20G syringe needle and, visualizing the DNA bands, stab the desired band of interest 2-3 times. No need to pick up any gel – just stab the band as you see it.

4. Next, I swirl the needle in my new reaction for a few seconds, then cap the tube.

5. Then I run the PCR amplification again using the same reaction, but only 20 cycles this time.

That’s it! You should then get a nice clean band on the next gel corresponsing to the one you stabbed with the needle (I run some of the previous reaction alongside).

One band from many and one of my favorite techniques. Its not going to help your real-time PCR :-) but great for things like ITS primers , which may cross react with plant species.

If you try this, let us know how it works. Do you have any other techniques for rescuing a poor PCR reaction?

Reference

Bjourson AJ, Cooper JE. (1992). Band-stab PCR: a simple technique for the purification of individual PCR products. Nucleic Acids Res. 20(17):4675.

more, more plasmid

Fri, 01 Oct 2010 12:54:34 +1300

In the days when doing plasmid minipreps, I certainly enjoyed the increased yields from using Terrific Broth (TB) over standard LB. Although, because it took a while to make - and would get pinched by others and inevitably contaminated - it was always labelled as something else. UFB used to stay on the shelf quite happily. Indeed, if the recipe was for Terrific Broth then mine was better than just terrific - it was Un-%&$#ing-Believable.

*cough* yes, well - that was some years ago

I was reminded of this when sending out a reference recently - on a newer broth reported in 2007, designed specifically for increasing culture density and plasmid yield. This newer media (PDM) includes magnesium and ammonium chloride and reportedly gives twice the yield of plasmid DNA than cultures grown in TB.

Feel free to request a sample of one of our plasmid kits to try this method on - the paper link is below and may you have unbelievable luck with the media.

A week of QMB

Tue, 07 Sep 2010 11:30:36 +1200

9 meetings, 6 days, 1 earthquake on leaving. Departure day for many of the attendees was marked by the large quake in Canterbury (felt in Queenstown) and our thoughts are with those scientists and friends in the affected areas.

This year was the first year for dnature as a trade exhibitor - many thanks to everyone who came by the stand/entered competitions/talked qPCR and high resolution melting analysis (HRMA). As I was talking in the MapNet meeting about HRMA it was great to see the number of people now using this method for various SNP and mapping applications.

On our stand we featured the Eco qPCR instrument - a lot of interest for both qPCR and HRMA users as well. To tie in with the 'eco' theme we featured the prize of top NZ wine from biodynamic (or 'eco' - sorry James!) producer Millton here in Gisborne. Congratulations to Phillip Wilcox from Scion in Rotorua who won the prize. For those that want to see more about the wine, check out www.millton.co.nz. I recommend the Viognier - a variety we discovered on moving to Gisborne.

For those that couldn't be at QMB this year, you can see a small part of it at the QMB Facebook page . . .Matthew Barnett's 20th anniversary song to QMB. Fashionomics, anthem. . . .what's coming next year? Another Queenstown Research Week, that's what. Thanks to Peter Shepherd and team, Dinamics team for a mind-boggling juggle of venues and registrations and look forward to seeing you at QRW next year.

Andrew Szentirmay from Australia on the dnature stand.

The dreaded 'c' in PCR

Wed, 18 Aug 2010 21:43:17 +1200

Well, its probably the same as tempting fate when mentioning 'the Scottish play' . . .but then we're not overly theatrical so we can say MacBeth. However superstition may reign when you get the band on the additional lane on the gel or the extra qPCR amplification curve and think 'did I load template in that reaction?'. That's before the repeat reactions tell you that it's con . . .contam. . .the dreaded 'c'.
Thankfully instances of it are usually rare in real-time qPCR (hopefully only the one gel to analyse products when establishing an assay) but it can be a major hassle to eliminate.
One of the difficulties can be the research desire to know where the contamination is coming from versus immediately getting back to strong positives and clean negatives. In the former, often one reagent may be exchanged at a time - often in the vain attempt to avoid throwing out enzyme vials or master mix, while spreading the contamination further.

Ditch everything

The fastest and ultimately the cheapest way to get rid of the contamination. There's often the urge to put the old reagents into quarantine to test later. . .but ditch them now since really, you know you'll never go back to them. Clean your master mix set-up and template addition areas with 10% bleach, followed by 70% ethanol and wipe your pipettes with the same regime. Other agents are often used as sterilisers (e.g. 3% hydrogen peroxide) but a quick wipe with ethanol doesn't cut it. Break out the newly-washed labcoats as well.
As a habit I always make up primer aliquots or reconstitute new primers the first thing in the morning, before working with any DNA samples. Does it prevent anything? I don't know, it's just a habit I've always used. So I suggest spend time in the remainder of the day for cleaning the areas and your equipment and then make up new primer aliquots the next morning (if you're working with a set of primers frequently, it's worth making multiple aliquots to avoid dipping into your stocks too often).
For more information on PCR contamination, it's always worth re-reading the original paper on the topic.
Avoiding false positives with PCR (1989). Kwok S. & Higuchi R. Nature 339, 237 - 238.

This has the great illustration of how many copies of potential contaminating template are waiting to ruin successive reactions. Dropping an amplified 100µl reaction into an Olympic-sized swimming pool (maybe swimming up and down a few lengths to mix it) and then taking your 100µl out again would give 400 copies of amplifable template.

This article was a waste of time I'm sure and you never see anything in your negative lanes - but add a comment if you have your own methods of dealing with 'c'. . . .or what it took to overcome an incident.

The solution (of your primers)

Fri, 30 Jul 2010 16:59:09 +1200

As PCR primers become less expensive, is there less care being paid to them? Throw in a diluent,a quick vortex and dilute for PCR?
I hope this doesn't sound too familiar because even in these days of real-time qPCR and various probe chemistries, your PCR primers still reign king in the reaction. Your probe design is not going to determine the efficiency and - usually - the sensitivity of your PCR and qPCR reactions. Therefore its worth paying a little care in looking after your PCR primers - especially if they're being used for a large number of runs and samples.

upon receiving them, briefly centrifuge to bring the lyophilised contents to the bottom.

To make up a stock concn of 100µM, take the number of nanomoles of primer delivered from the tube and shift the decimal point to the right, to get the number of µl to add. For example, from a recent design I ordered from Biosearch (whom we now distribute but I digress), I received 44.23nmol of primer (50nmol synthesis scale). To make my stock solution, simply add 442µl diluent.

The best diluent? It seems to be a personal preference for everyone. I like to use TE' (aka TE0.1) where the EDTA is at 1/10th the standard concentration normally in TE buffer. So that's 10mM Tris, 0.1mM EDTA (pH8).

After adding the diluent, I leave the tube(s) on the bench during coffee ah, for 10-15 minutes, a brief vortex, pulse centrifuge and I can make my working dilutions (typically 10µM).

Some suggest to make a more concentrated solution that can then be checked on a nanodrop to ensure accurate concentration determination but I've never had an issue with the amounts received.

If you have some suggestions on looking after your primers then please add a comment or e-mail us

A gene by any other name

Tue, 13 Jul 2010 19:09:25 +1200

Nothing displays a scientist's sense of humour (typically bone dry and sardonic) like the names they give to genes. This article was prompted by seeing the paper again with my favourite gene name (see later). Some other great names out there:

RING

SUNDAY DRIVER

JAK

INDY

KEN AND BARBIE

MAGGIE

SWISS CHEESE

TINMAN

GRIM and REAPER

JORDAN

Really Interesting New Gene

intefering with intracellular traffic (Drosophila)

mutations in this gene involved with leukaemias, it was published as Janus kinase
but apparently the original expansion was 'Just Another Kinase'.

'I'm Not Dead Yet' (courtesy of Monty Python) - Drosophila with this gene mutated
live longer

Drosophila male and female mutants lack external genitalia (you're checking your
kid's dolls aren't you)

arrests development in Drosophila (similar arrested development in Maggie
- the youngest - on the Simpsons)

Flies have holes in their brains like. . .well, you get the idea

Drosphila embryos have no heart

work together to mediate cell death

an extremely mobile element in an algae species, able to jump with great ability around the genome

My favourite gene name though? A gene that helps maintain the timing of arabidopsis circadian clocks: TIME FOR COFFEE. Speaking of which. . .

Feel free to post a comment on your favourite gene names that you've come across

(Thanks to curioustaxonomy.net and CleverGeneNames for many of these names)

Your resolution to melt

Mon, 28 Jun 2010 11:13:42 +1200

Amplify them all and let the melting sort ‘em out.
OK perhaps my attitude to analysing sequence variation may be a little flippant but it characterises a lot of my thoughts toward High Resolution Melting Analysis (HRMA).

It is now more than a decade since melting analysis to characterise PCR products was introduced. This technique, typically after SYBR Green-based real-time qPCR, is an economic and flexible mainstay of research laboratories around the world for applications such as gene expression due to their ease of design and reduced cost (just primers, no labeled probe(s) required).

In recent years, the advancements in dyes for qPCR, instrument heating precision and new software algorithms are now doing the same for sequence variation analysis – meaning high resolution melting analysis is becoming widely-established as a rapid, cost-effective and far-ranging means for rapid sequence analysis.

Originally described for SNP variant analysis (and still the leading application), HRMA is now being used in a wider context such as HLA comparisons, microsatellite genotyping and methylation status of DNA sequences. Even within SNP analysis, HRMA may be applied in several ways – scanning regions for heterozygotes (and possibly homozygous variants) or for genotyping a known SNP. Newer developments such as unlabeled probes and snapback elements on PCR primers allow the simultaneous genotyping of a desired SNP along with the scanning of the amplicon for any other sequence variation.

New methods and new applications means then exponential trend of HRMA publications is unlikely to plateau for quite some time. Get melting!

You may wish to view:

‘ HRMA – beyond the SNP’ (webinar on www.bitesizebio.com/seminars). Webinar on 22 June, 2010 - recorded.

Suggested reading

Montgomery JL, Sanford LN, Wittwer CT. High-resolution DNA melting analysis in clinical research and diagnostics. Expert Rev. Mol. Diagn. 2010;10:219-40.
Erali M, Wittwer CT. High resolution melting analysis for gene scanning. Methods 2010;50:250-61.
. . . anything else by CT Wittwer
Vossen RH, Aten E, Roos A, den Dunnen JT. High-resolution melting analysis (HRMA): more than just sequence variant screening. Hum. Mutat. 2009;30:860-6.
Highly recommended review describing a number of applications for HRMA

If you're doing HRMA already, please add a comment with your application - always great to hear new ideas