Getting the most off your PCR columns

Posted by John on 20 May 2010 | 0 Comments

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A quick tech tip I've found useful to maximise yield if using a low elution volume for column purification ie <50µl. I usually use this for PCR reaction purification but it also works for genomic DNA and probably other extractions as well (e.g. plasmid and RNA) if you're using lower elution volumes than suggested.

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Molecular Diagnostic Workshop part 2

Posted by John on 16 May 2010 | 0 Comments

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Continuing on the 2-day molecular diagnostic workshop at MAF NCBID (Part 1 here):

A discrete PCR assay allows us to check for a pathogen of interest - but when an outbreak is an unknown agent? Richard Hall (ESR) covered new technologies designed to identify these viruses and bacteria, as well as their collaboration with the developers of the technologies at the Centre (OK, its American, we'll call it Center) for Infection and Immunity in New York. MassTag PCR combines high level multiplexing with the resolution of small chemical tags on the 5' end of the primers that can be resolved via mass-spectrometry. No worries about the effects of amplicon size competition or limits of gel resolution/fluorophore wavelengths. Richard also covered the GreeneChip - a microarray designed to detect and one (or more) of thousands of pathogens detailed on the oligo chip. Richard and an ESR team are looking to bring these technologies into NZ - including the use of next-gen sequencing that was also described by Chris Pope (ESR) for surveying the microbiome of the cystic fibrosis lung.

I was grateful and heartened by the feedback received on my own talk the following morning - jumping around a bit from multiplex TaqMan detection of grapevine viruses to the use of new HRMA forms for human SNPs. I could have jumped around more. . .
Detecting a range of RNA virus isolated in grapevine for my own work has been shown to be tricky at times due to the potential (and very real cases) of false negatives due to sequence variation. On the other side of the coin, a very timely talk was given on the issue of false positives by Richard Spence (MAF IDC). A spate of positive tests for a bovine bacterial pathogen involved an international team before a published qPCR assay was found to be using a target region found in multiple species - a timely reminder for the on-going requirement to BLAST primer and probe sequences for the possibility of both issues - false positives and false negatives.

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Molecular Diagnostic Workshop

Posted by John on 9 May 2010 | 0 Comments

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Last week, MAF IDC (Investigation and Diagnostic Centre) & NCBID (National Centre for Biosecurity and Infectious Disease) held a two day molecular diagnostic workshop. I was fortunate to be invited as a plenary speaker and was pleased to catch up with a good number of people at the meeting - as well as meeting some new researchers in this area.
As the meeting opening, I had given thought to trying to provide the odd update on Twitter during the meeting. As I mucked around with my phone I realised I'd rather be listening than tapping on a phone and so that idea went straight out the window. The meeting was opened by IDC Acting Director Dr Joseph O'Keefe who described how the molecular area had become a mainstay of diagnostics.
The opening plenary was given by Prof Nigel French who gave a good overview of molecular use in the epidemiology of a number of food pathogens. This molecular epidemiology and isolate typing was to be a common theme of the meeting with talks from scientists at the Hopkirk Research Institute and ESR.

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