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.

Grrrrr.

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

micropipettes

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….

gels

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

Interpreting your Chloroplast DNA PCR results May 2018

Synoptictastic!

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