It’s no secret that qPCR is my bag; it’s been almost my sole focus since the summer of 2007. I am not, by any stretch of the imagination, an expert but over the years I’ve come to pick up some tips and tricks to help me produce good data and I thought I’d share:
Look after your prep area
It may seem a little OTT, but I treat the area in which I prepare my qPCR experiment in the same way I treat a laminar flow cabinet in which I’m culturing cells. I learnt very early on that getting amplification in a qPCR reaction is easy, it’s the not having amplification which is hard. I prepare my qPCRs on a big yellow tray. The tray is thoroughly cleaned with TriGene Advance and 70% IMS and then anything and everything that touches the tray will also be cleaned. This way I know where my clean area is and can keep my sensitive samples away from the relative filth of the lab bench. My gloves (and lab coat sleeves) are regularly sprayed with 70% IMS and my pipettes are wiped down with an IMS soaked cloth between each use.
Get to know your pipettes
Micropipettes can play up for a number of reasons and if you’re in a situation where you’re sharing pipettes, you may not be away of any mishaps that might have happened with them. When you’re pipetting only microlitres at a time, even the smallest variation can make a big difference. Calibrating your pipettes regularly is very important but in between times get to know what 1µl looks like in your tips and make sure you take a look at it each time you dispense. You’ll soon be able to notice if something isn’t quite right. Accuracy is key with qPCR; in an ideal world a robot would set up our qPCRs while us inaccurate scientists might just about be trusted to press the buttons…but the robots would probably just do that too.
Have a routine
Have you ever added one primer into your master mix, gone to add the second and not been able to remember which one you’d already done? I always, always, ALWAYS add my forward primer before my reverse, that way, when I find myself in the above situation I know which one to add. This has saved me from having to start over on a number of occasions.
Awareness in any scientific experiment is a good thing. Your reagents aren’t going to tell you when they’re unhappy so it’s important to know what they’re sensitive to. SYBR Green doesn’t like light so keep it in the dark as much as possible. No qPCR reagents are going to be happy with heat so keep everything on ice unless absolutely necessary. Knowing your reagents’ likes and dislikes will help you get the best from your experiments.
On a side note…
It seems that paranoia is a recurring theme in my posts. It’s probably because even the smallest of mishaps in science can set your research back a very long way, be it by infections in your cultures, failing freezers or, as I’ve learnt this week, burglary.
A friend of mine is currently writing up her PhD at a university in the south of the UK. I imagine she’s experiencing the highs, lows and periods of extreme boredom that come along with staring at a computer screen all day. In her own words she had “got sloppy in backing up” and hadn’t saved her progress anywhere but her laptop in six weeks. Her flat was broken into and her laptop stolen.
“It was heart breaking losing so much work!” she told me “back up all the time when you’re writing. Having to do it again isn’t fun! I know having your laptop stolen seems unlikely but it’s better to be safe!”.
I can’t imagine that being burgled is pleasant but losing a month and a half’s work doesn’t bear thinking about.
Just a little reminder to you all: Back up your work every day!
Karly first joined BJS Biotechnologies as an undergraduate placement student in 2007. A bachelor’s and master’s degree later and Karly is back working with BJS but this time as a PhD student in conjunction with her alma mater, Brunel University. Her research looks at the mTOR signalling pathway in endometriosis and ovarian cancer.
In her spare time, Karly is learning to play the guitar and drinks a lot of tea.
You can follow Karly on twitter @KarlyBroadway