Tales of PCR

Ten long years ago, when I first started work at OHS, I was chatting with a father of a student at a 6th form parents’ evening. It turned out that he was in charge of a small Biotechnology company which was closing down. I’m often a bit slow on the uptake, but even I couldn’t miss this open goal.

“What happens to all the kit?” I said, casually.

“Oh it just gets chucked out,” he said, cheerfully.

“Um, if you don’t want it,” I said, “can I have it…?”

And so it came to pass that I borrowed a friend’s large estate and drove down to an industrial estate near Abingdon and filled the back with all kinds of biotech goodies. I made off with Gilson micro-pipettes, a massive stash of tips and micro-tubes that we’re still using, 10 years on, and, treasure of treasures, a thermal cycler….

thermal cycler

Oh how I loved it!!! It was sleek and chunky and alluring. It looked amazing, it felt amazing, it just shouted, “Serious Biology.”

Only problem was, I didn’t have a clue how to use it. And while it did come with an instruction manual, it was not written in a language I could understand…

So for the next 2 to 3 years, my lesson on PCR consisted of me bringing the thermal cycler out of the prep room, fondling it, opening and shutting the lid with a satisfying clunk, whilst explaining how it worked with a little bit on Kary Mullis thrown in for good measure.

And so things would have probably continued until…

…  I found myself chatting to another parent who was a Professor in the BioScience department at the university. Parents can be very useful…. Somehow, our PCR machine came up and my inability to use it, so she immediately offered to send one of her post-grad students over to explain how it all worked.

Which he did. He also commented that it was a far better machine than the one owned by his department. Hurrah! This moved things on a little bit – though programming the brute remained problematic as a) the screen was almost unreadable (you had to be in dim light and at an angle of 38′ to make out the text) and b)the memory function wasn’t working. It would take me 20 minutes to plug in all the details, but unless you ran the programme immediately, all the instructions would be lost.


While all this was going on, I had been investing heavily in biotech kit. Lots of Gilsen micro-pipettes with a range of volumes.


Powerpacks, micro-centrifuges, vortexers, gel tanks, visualisers…. we are now seriously well-equipped!

biotech stuff

And it gets used. We transform E.coli and separate proteins with electophoresis and have tried a whole variety of DNA analysis kits. But I was desperate to do PCR.

The problem was identifying a workable protocol from which the students could not only learn all the relevant skills, but also which reliably produced interesting and usable data….

None of them worked. We tried a kit that amplifed non-coding Alu insertions from cheek cell DNA…. would have been brilliant for all kinds of synoptic stuff, from interpreting the gel to evaluating Hardy Weinberg distribution, but we got not a sausage, just some faint primer dimers on the gel front.

We tried a kit that amplified alleles of the PTC taste receptor protein, also from cheek cells. This would have been glorious – linking phenotype to genotype in the most direct way imaginable and really testing understanding of all the key concepts. Again, nothing.

I even (briefly) looked into emulating my old school where they (amazingly) use PCR to carry out site-directed mutagenesis on bacterial plasmids, but I just don’t have the background or the experiences or the training to make this remotely viable. After all, if I can’t get a ready made kit to work….

It was incredibly frustrating! And expensive (these kits don’t come cheap). I consulted friends and colleagues and contacts and the suppliers, but all to no avail.

In frustration with our inherited thermal cycler, I bought a new one… would this solve our problems…?

new thermal cycler

This had major advantages over the original – you can read the display, it’s very easy to programme, it remembers the programme…. and it’s PURPLE!!!!

But could we get PCR product? Could we buffalo…

And then…

We finally cracked PCR this year. The NCBE produces a PCR kit

for amplifying non -coding regions of chloroplast DNA. It’s based on FTA cards where you squash a leaf of your chosen species between two bits of card. Leave it to dry and then punch out a disc which is used as the basis of the PCR. I trialled it with some Year 12s last summer and rolled it out in full for the Year 13s this year.

We’ve had 100% success. Which means that in addition to the satisfaction of actually getting observable bands on your gel, the students also get to interpret the results and use them to construct possible phylogenies of a whole range of plant species.

Here are some recent results….


Along with an exercise in figuring out what’s going on…

Interpreting your Chloroplast DNA PCR results May 2018



Here’s my horse’s leg!

So having acquired a horse’s leg, what do you do with it?

Ignoring some of the cruder suggestions that I know some of you are thinking (yes, I mean you Bill), here’s how I use it in lessons with my Year 11s/12s…

I start the lesson with the story I wrote last week. It’s a good introduction for what follows, if only because it grabs their attention. Huh? here’s this going?

I then ask, what have we got in common with horses?

It doesn’t take much prompting to unleash a deluge of similarities – we’re both animals, both mammals, have two eyes and ears, a mouth, a nervous system. We have a digestive system and a back bone…

Ah! Yes, both vertebrates. And, in fact, both possessors of a fine skeleton.

What do all these points tell us about our respective family trees?

Again, they’re very happy with the idea that at some point in the past, we shared a common ancestor. Which is why we both have, for example, legs….

I fish out the first bone. It’s a whopper. Which leg?I ask.

For some reason, they always think it’s the back leg. But no, it’s actually the front.

Which brings me to the Curious Incident of the Badger in the Night. Because ideally I would not start with the enormous horse humerus, but the horse scapula, because it’s so glaringly obvious what it is and so glaringly similar to our own scapula, that great flat sheet of bone for powerful muscle insertion. It’s perfect for setting the stage of homology, which is of course where this is all heading, as it establishes more clearly than any picture or discussion that their bones are our bones.

So why don’t I have one?

Well, I did. The leg so generously deposited on our drive all those years ago was in full possession of a scapula, and a scapula was certainly deposited, along with all the other bones, into the dustbin of horse-stock that I described last week. But at some point in the Neutrase X-tremo-Stench digestion that followed, some wretched animal –that must have found the smell appealing… – found the dustbin, nosed off the lid, and made off with the top-most bone, a nice juicy scapula. A badger? The neighbours’ dog? We’ll never know. And I never found it, despite extensive searches. Grrrrr….

Still, it adds another nice little detail to the story – they think it’s funny – and we’ve established, via a rather roundabout route, that this is a horse front leg. We’re also standing next to the lab human skeleton.

OK, if this bone was attached to a horse scapula, what is it? What’s the equivalent bone in a human?

Yes, that’s right, it’s a humerus. How are they different? And why?

horse leg 1

It’s a striking contrast. The two bones are pretty much the same length, but the horse one is massively thicker and heavier. Of course it is. Horses don’t use their front limbs for swinging through the trees or picking fruit or grooming other horses. They need load-bearing supports that can hold up half a tonne of equine beast.

OK, if this is a humerus, what’s next? They generally know their skeletal anatomy – yes, we should have a radius and ulna.

Quick aside – which one’s which? This they don’t know. Or just guess. There are groans for my handy aide-memoire – the ulna articulates with the ulbow, while the radius articulates with the rist.

horse leg 4

So what’s going on here? Because the next horse bone seems to bear no resemblance whatsoever to the human equivalent. It appears to be a single, massive load-bearing bone again.

horse leg 7

But wait, look more closely. There are two bones, one articulating with the ulbow, the other with the rist, but they have fused together. It’s a beautiful illustration of the line from the Evo-Devo song – “safer the mutation aimed at regulation, keep the building blocks but switch their activation…”

It’s almost my favourite moment. As evidence for a shared ancestor, it’s compelling. As evidence for evolution itself, it’s hard to beat. No divine creator would make such a bodge job if designing the thing from scratch. The students think it’s seriously cool. Look! A horse embryo develops exactly the same bones that we do, and then glues them together because it actually only needs one, and for a completely different function.

But it gets better. If the broad base to this bone makes it the radius, what bone should come next? A quick cross-check with the human skeleton, and they realise it’s the wrist, not just one bone, but lots, the carpals.

horse leg 2

And here they are, a really pleasing set of bones with an intriguing jigsaw puzzle for anyone sufficiently interested and motivated to try and piece them together.

But how odd! The horse’s wrist is half way up its leg. Again, only evolution can explain the idiocy of having a cluster of blobby bones half way up a load bearing limb. Intelligent design? Give me a break…

On we go. What’s next? The finger bones! The meta-carpals! Again, something seriously weird is going on.

horse leg 9

There only seems to be one meta-carpal, but look closely and there are the others, shrunk and withered and, again, fused either side of the central one.

horse leg 8

We put them out in order, ending with the pleasingly shaped “hoof-secreting” bone.

horse leg 3

horse leg 6

And I ask them to demonstrate their understanding by giving me a simply visual indication of what a horse runs around on. … Yes, that’s right. It’s their one and only chance at school to safely give their teacher the finger…

Where’s your horse’s leg?

When I first became Head of Biology at Oxford High, I had a poke around the department and eventually had to ask:

Where’s your horse’s leg?”

“We don’t have a horse’s leg.”

“What do you mean, you don’t have a horse’s leg? You must have a horse’s leg! You can’t teach biology without a horse’s leg! Where is it?”

“We don’t have a horse’s leg.”

“You don’t have a horse’s leg?!?!?!?!?”

“We don’t have a horse’s leg.”

I was shocked. It seemed that they didn’t have a horse’s leg.

Oh well, I thought, that’s quickly rectified, and I started hunting on line for horse legs. By which I mean the bones of a horse leg. This proved harder than I anticipated. None of the educational supply companies stocked horse legs. I eventually found one company that could supply an entire horse skeleton for about £3000, but nothing remotely resembling a single horse leg. Ebay, Amazon, horsesleg.com, all came up short.

I rang the Natural History Museum in Oxford. They were lovely!

“Have you got a horse’s leg?”

“Yes, we’ve got lots.”


“Can I have one?”

“Sure. To borrow.”

“Oh. I want to keep it. Can I have it on permanent loan?”


Damn! It was back to the drawing board.

So I had a think, and decided to track down people who made a living preparing specimens for museums. There aren’t that many… But I eventually made contact with a chap who had lots of good advice. I would need a container for the specimen that could be filled with water and heated – a saucepan or similar. I would need a thermostatic heater. I would need water. And I would need Neutrase


a powerful protease that works best in neutral (hence name) conditions…

Oh, and I would need a horse’s leg.

This last detail seemed a bit of an obstacle, but it proved far easier to source than I imagined.

New to the Oxfordshire area, we had been invited to a lunch party to mingle with local folk and maybe make some friends. I ended up chatting to a man who described himself as a Trainer and Breaker. Of what? Why, of horses, of courses….

Ta da!!!!!!

Well into the second bottle of lunchtime champagne, I said casually,

“these horses you break and train, do they ever, um, die….?”

“Yes, all the time. They break a leg or get an infection or can’t run anymore and they’re put down.”

“Um, the next time, um, one of your horses dies, um….”


“Can I have a leg?”

He was brilliant. He didn’t bat an eyelid. Yes, of course I could have a leg. No problem at all. Front or back?

I told him my preference, gave him my contact details and we parted on good terms.

The very next morning the phone went at about 6am.

“Were you serious about wanting a horse leg?”

“Yes, absolutely!”

“Because we’ve just had one die. Shall I drop it off later this morning?”

“Yes please!”

Long pause.

“It was rather a big horse…”

I didn’t want to let the moment slip.

“I don’t mind! I’ll take it!”

And so just a little later that morning, my wife opened the curtains to look down at our yard, to see a scene like something from the Godfather – a large hessian sack, containing a freshly hacked off horse leg, bleeding profusely into the cobblestones…

He hadn’t exaggerated. It was huge! Nearly as tall as me, and I’m only just under 6′. It must have been a monstrous great thing.

But, key question, how do you set about dealing with a freshly severed horse leg?

First step, remove as much of the flesh as possible. I was sent to the bottom of the garden where I was able to suspend the leg from a tree with bailer twine and use the large kitchen knife to carve off the skin and muscle. This was pretty gruesome and I filled a wheelbarrow with horse meat. A nearby sett of badgers enjoyed a free feast that night…

I now had lots of bones, still held together with scraps of connective tissue and all covered with small bits of meat and tendon. I disarticulated the main bones and put them all into a large metal dustbin I had bought for the purpose. In the larger bones, I drilled a couple of holes…. I then built a little bonfire under the dustbin and heated it to just below boiling. This melted all the fat, particularly the marrow fat which drained from the holes, and formed a disgusting scum at the top of the dustbin….

I dug a hole in the ground to pour the waste water into, refilled the dustbin with fresh water, added the Neutrase and inserted the thermostat, set for 40’C…. and left it for a week.

Oh lordy lordy the SMELL! It was AWFUL, gut wrenchingly HORRIBLE, quite the most disgusting olfactory experience I have encountered. The resulting dustbin soup resembled something they might serve in Hades.

But after a week, as per instructions, I was able to pour off this liquid to be left with…

my long awaited horse leg bones!

horse leg bones

Now why, the patient reader might be wondering, had I gone to all this trouble? What is the educational value of a horse leg?

For that, you’ll have to tune into my next post where I’ll describe that lesson … along with the Tale of the Curious Incident of the Missing Scapula….

thanks for reading!

Whipping up a storm

I’ve been out of the burble-sphere for a while as it’s been a busy term and I’ve been fully occupied with other things, principally two new projects that I’m setting up in school. One, a bee keeping club, is currently in the hive building stage and there’s nothing very exciting to report yet.

But the other is proving to be one of the most fun, interesting, challenging and exciting things I’ve been involved with at school…. and yet it nearly didn’t happen!

It started at a conference I went to in Birmingham last year. One of the other speakers described this amazing project where A-level students were doing original molecular research into Multiple Sclerosis. I was stunned. And immediately wanted to set up something similar. But despite attending the IRIS symposium at the Wellcome Trust HQ in London – amazing building – and finding a small group of students desperate to carry out this kind of research – we could not locate a single research group in Oxford who were interested in this kind of collaboration.

But then, at another conference this October – a school science conference in Tonbridge – I ran into Becky Parker again and she started enthusing about their Whipworm Genome project. I wasn’t immediately sold – it wasn’t the kind of hands-on, micro-pipetting/PCR-ing/gel electrophoresing/fluorescent visualising that I knew my students craved. Sitting in front of a computer scanning a genome for possible genes? I couldn’t see the appeal…

But she took my details, put me in touch with the head honcho at the Whipworm Genome HQ, and I signed up, thinking I might get 5 or 6 of the best Year 13 students interested.

Got that wrong.

I gave an assembly outlining this horrible Neglected Tropical Disease (neglected because only poor people get it, and who cares about them?). I described the project – bioinformatics/genomics – and what it would involve.

Whipworm Genome intro

Note the last slide – a screenshot from the Apollo software involved. I told them not to panic – it was as much a mystery to me as it was to them. I told them we would figure it out together, that I fully expected them to be teaching me, just as my 11 year old son schools me on how to play Clash of Clans….

And something struck a chord. A combination, I think, of:

  • genuine, original research – when they look at a whipworm gene, they are the first person to ever examine it
  • cutting edge technology – gene curating of whole organism genomes is Where It’s At…
  • a challenging and very steep learning curve (more on this in a mo!)
  • a worthwhile project – this might actually make a difference – it’s got immediate relevance
  • it’s online software, so you can do it anywhere you have a computer and internet access
  • looks good on CV/personal statement
  • immediately relevant to A-level…

Whatever it was, I currently have 86 girls signed up. I fully expect some of these to drop out, but not many. The interest and motivation is fantastic.

And it’s been a brilliant learning experience for me. I’ve never analysed a genome before, never curated a gene, never used the Apollo software. So having to do all that, whilst simultaneously trying to figure out how to teach it to students who currently don’t know a huge amount about DNA, has been hugely satisfying.

I started them off with a crash course on DNA

DNA intro for Whipworm Project

– the key bits for this project are understanding the concept of an anti-parallel 5′-3′ double helix; knowing about base pairs; knowing about introns and exons (or CDSs); knowing about UTRs (new to me!); knowing the identify of START codons and STOP codons; understanding that DNA gives rise to RNA which is translated into protein.

That’s a lot to take in for students who don’t know much about the basics of DNA!

But even being familiar with this doesn’t make the Apollo on-line software immediately accessible. So I had to practise with lots of examples and slowly build up a sense of how to do it, what to look for, how to trim or lengthen or adjust the computer gene predictions to what actually matches the evidence.

Because this is the really interesting bit. There are sequences that a computer has identified as looking a bit like a gene. But the computers often get it wrong. The student then has to compare the computer predictions to other data (principally RNA sequence data) derived from living whipworm cells, to see if the prediction matches what’s actually going on in real life…. and then adjust the predicted gene accordingly.

It’s fascinating, compelling and extraordinary. The power of the software, the sheer quantity of data, the elegance of the program, are all strongly addictive. At the moment, we’re just trying it out, running through a section on Chromosome 1 (whipworms have 3 chromosomes) for quality control issues. But just before Xmas, we should get our very own unique section of Whipworm Genome to annotate. Can’t wait!!!


I’ve burbled on this topic before, but only briefly, tucking it at the end of a piece principally about World War 1 Shell Casings and the link to deadly diet drug, DNP…. I want to revisit it, primarily to describe in more detail the lesson, how I structure it, why it works, and use it as an example of how you can turn a non-practical lesson into a process of inquiry and discovery.

See what you think. Better still, let me know what you think!

Year 12. We’ve just about finished Cell Ultra-structure, they’re about to do a homework on Electron Microscopes. I show them this picture…


As ever, Powerpoint for me is a way of projecting thought-provoking images or interactive animations. I never use it to deliver notes – the phrase “Death by Powerpoint” exists for a reason…

So, what is this?

And immediately, they’re engaged. It’s a mystery! A puzzle! They’re all thinking and all have some kind of suggestion. I also use it as a way of encouraging involvement – have a go! what’s the worst thing that can happen? you might be wrong! but at least you tried…

After a round or two of interesting ideas, I start providing clues and someone usually guesses, correctly, that it’s a human embryo on the end of a pin.

I follow this up with another picture of a human embryo, just a bit closer in.

embryo 1

OK, key question, what did this start life as?

Yes, that’s right, a zygote. A single cell. Formed when a sperm and egg fuse…

But that egg, where did that come from?

From the mother. Of course.

And how did that individual start life? Yes, exactly, another zygote! Also formed from a sperm and an egg…

And that egg, where did that come from…?

We start a regress, back through the generation, through ma to grandma to great grandma and beyond.

If we keep going, I ask, where do we eventually get to?

To apes, someone say! Well, OK, yes, a common ancestor of us and apes to be strictly accurate, but no, much further… how far can you go?

They get it. The very first cell… This year, one of them can actually refer to LUCA (Last Universal Common Ancestor).

OK, all very cool and a bit mind boggling and stuff, but how would they describe that very very early cell, in just one word?

We try out a few suggestions before agreeing on “Simple”. This makes sense. Simple things evolve before complicated things. But what does that actually imply? What would this very early, very simple cell, have looked like?

We can’t know, of course, but as a way of stimulating discussion, I show them this:


What do they notice about this cell, particularly compared to the cells they’ve been studying?

That’s right – it’s got no nucleus, no mitochondria, no rough endoplasmic reticulum, none of the complicated internal structures of the rat liver cell that has cropped up so frequently on their interpretive electron micrographs.

So if this isn’t LUCA, what is it? What very simple cells are still among us? Again, they generally get this – it’s a bacterium.

I then contrast it with a more familiar A-level cell…

plant cell

… from which they can cheerfully pick out half a dozen different clearly visible structures.

Which brings us neatly to the key question of the lesson. How did life on earth go from this…


to this…

plant cell                           ?

At this point, I construct a time line on the board, starting with the origin of the Earth 4.5 billion years ago and sign posting it with the key relevant events along with the way (origin of life, 3.5 billion years ago, origin of complicated cells, 1.5 billion years ago, origin of modern humans, 200,000 years ago…). I then use this to add a few notes about the types of cell involved – the simple ones, the Prokaryotes, and what this means, and then the complicated ones, from which all multi-cellular life evolved, the Eukaryotes.

This allows us all to draw breath, before we return to the key question…


We bat a few ideas around. I drop a few visual hints (mainly be juxtaposing the two images and asking them what the bacterium resembles). And eventually someone has the necessary brainwave – did one of them start living inside the other?

Bingo! Endosymbiosis! Lynne Margulis’ brilliant idea that was, as things so often are, ridiculed and rejected….

Lynn Margulis and Endosymbiosis

And finally we can get on to the main exercise where I get them to figure it out for themselves (see attachment above). For if mitochondria used to be free-living bacteria, then perhaps we can make some predictions about what we might expect mitochondria to be like.

I put them into groups of 4 and get them to jot down all the things they already know about bacteria, without looking anything up in their books or their smartphones. Their list will eventually include at least some of the following…

  • they’re really small
  • they have plasmids
  • they reproduce through binary fission
  • they have ribosomes
  • we kill them with antibiotics

From this, I get them to come up with simple 5 predictions about mitochondria, again without looking anything up. Things like, “if mitochondria used to be bacteria, then they should have ribosomes…”

Once they have a list of 5, I let them check their predictions. And lo and behold, they’re right! There is evidence for endosymbiosis, even if it happened over a billion years ago. And they figured it out.

They’re convinced. They’re happy. It’s a successful lesson.

But now contrast it with an alternative lesson plan, perhaps put into a Powerpoint presentation… it might go something like…

“Today we are going to learn about endosymbiosis. Endosymbiosis is the theory that cell structures like mitochondria used to be free-living bacteria. Evidence for this theory comes in the similarities between the two. See the following list…”

It’s the self-evaluation every teacher should carry out before every single lesson plan – am I teaching? Or am I delivering information? The difference, at every level, could not be greater…

Russian roulette…

Just a quickie this week….

It’s Year 10 and the grim effects of smoking. The biology is interesting, though I must admit I prefer to celebrate the power of data to do good things (Richard Doll) and get them thinking about experimental design (how to show a causal link between smoking and cancer?). The following exercise is designed to cover these learning outcomes and doesn’t need much comment from me (though do look at the wonderful quote from James 1st/6th!).

Smoking and Epidemiology and Richard Doll November 2017

But how to convey the sense of genetic Russian roulette that is random mutation?

For this, I came up with the following interactive Powerpoint…

Random mutations in DNA interactive

The first slide just introduces chromosomes and we have a very basic chat about genetics and the idea of coded information.


A mutation, I explain, is just a random change to that information – the key point is, it could occur anywhere….

The second slide indicates 22 loci (the blue circles).

chromosomes 2

I ask a student to pick a blue circle, a locus. Any one they like. Clicking on the blue circle will, 21 times out of 23, take them to a slide saying:

Silent mutation

No effect no person

Clicking on the smiley face takes them back to the second slide, and the locus they selected magically disappears. We talk about the idea of silent mutations. Another student picks another circle, with, one hopes, the same effect.

But at some point, somebody will choose the bottom circle of the two on chromosome 7. Clicking on this circle takes them to this message:

Mutation to onco-gene

Uncontrolled cell division


So I hope they take home the idea of random mutations and that smoking is like playing Russian roulette with your genes, but also the idea that the more you smoke, the more you increase that risk.

Short and sweet – let me know what you think!

Festive Viral Fun

So, what kind of teacher are you?

When Year 11 start Infectious Disease, I always get them to build Jolly Christmas Viruses. There’s quite a variety of cut and stick models easily accessible on-line…

virus model

…and if you photocopy them on to lots of different coloured card and lay in industrial supplies of glue and glitter and pipe-cleaners, you can’t really go wrong.

I do insist that every virus be different. I show them a clip from Secret Life of the Cell (about 15 minutes in) where we see an adeno-virus gain access to the cell using its antigen and a cell receptor (this kind of hinting at the glorious complexity of A-level Biology is all part of my retention and recruitment plan).

And then I put on some salsa music and let them get on with it for an hour…

When they’re done, they attach a bit of cotton thread and we dangle them from the lab ceiling, where they quietly drizzle glitter on innocent students for the rest of the year…


Here’s a not-particularly-special bacteriophage in mid-dangle.

I’ve worked with teachers who utterly fail to see the point of this lesson. It addresses no learning outcomes, it delivers no information, it utterly fails to tick a single learning objective on the specification, it uses up valuable Copy The Notes From The Powerpoint time…. I think they panic because they feel out of control – the students are working autonomously, and this is an uncomfortable feeling if you’re not used to it.


It’s fun.

It’s different.

It’s memorable.

They will do wonderful things – one student produced a virus with a little portrait of Justin Bieber at the end of each antigen – the virus that causes Bieber Fever….

It gets them asking all kinds of interesting and relevant questions.

Because how can something so tiny (a virus to a football is the same as a football to the planet Earth…) and so simple (bad news wrapped in protein) make you ill? Discussion of rabies and smallpox and herpes and AIDS all follow naturally.

Which leads nicely to Immunology. Try my simplified animated Powerpoint as an introduction to Clonal Selection and Expansion…

Immune response