Here you will find the musings of John. Some of these were written almost a decade ago now but they remain relevant today. We welcome your feedback.
Multiple reactions to run, each with their own preferred annealing temperature? Grabbed two primers of different Tm’s to amplify a region and trying to work out the best program? Touchdown PCR to the rescue. One of the best things to come out of Australia, it uses decreasing annealing temperatures during the cycling until it ‘touches […]
Originally published in May, 2014 ‘”we were interested to see if we could do it from hair . . .and we were the only ones around doing PCR” So you might have read the blog entry about the visit to see the first thermalcycler “Mr Cycle” at the Smithsonian. You haven’t? Sorry, another old blog […]
Another old blog article – this one written originally in July 2010 : 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 […]
OK, confession time. I used to work some crazy hours in order to fit some library screening into a part time job while finishing my degree. No, that’s not the confession – more, when I put the PCR on I would sometimes look out the window and note the wind direction. Occasionally, very occasionally (*cough*) if the […]
Designing primers for your high resolution melting is easy enough. A quick run with some known genotypes …. and look for the melting curve differences among samples. Too simple. But what about the temperature-neutral SNP’s? The what? A number of SNP’s ( ~ 4% in the human genome) have no affect on the melting temperature […]
You’ve done the hard work – you’ve got good quality RNA. You’ve seen ribosomal bands on a gel (or *cough* Fragment Analyzer). A good RQN/RIN (if that’s what you go by) and everything’s great. Now to get rid of the DNA Quick check (perform a PCR directly on appropriate amount of RNA) and often there’s […]
Reagent contamination in PCR has long been documented – not only by operators but also from commercial sources long before those operators ever laid DNA-ridden hands on them. While contamination of some/many Taq polymerase enzymes with residual host E. coli DNA has had attention, little attention has been paid to the ubiquitous DNA extraction kits. […]