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That last little job before running your qPCR - no, not trying to remember which random sample you loaded in the wells; rather, centrifuging down the reactions to the bottom of the wells. This removes air bubbles and ensures all reaction components are combined.
Working with RNA viruses (and multiple isolate strains), sometimes it can be hard enough to get sufficiently conserved sequence for primers (and harder if designing a TaqMan or probe-based assay). However a recent example in our lab showed the benefit of designing multiple assays for the same target and comparing the performance across template dilutions.
Happy 2012 to everyone. Of course I relate most things to PCR stuff (well, this is a PCR blog) and this year marks 20 years since I first did a reaction. Which means it's 20 years since my first blank gel and 20 years since I amplified strange bands, wrong-sized bands - before hitting the conditions and getting it right.
Or at least for the positive control. . .
An interesting article on Allan Wilson (with the words used in this subject line) in the latest issue of NZ Listener - promoting the upcoming lecture tour by the PhD student who, with Wilson, published on "mitochondrial Eve" - our common female ancestor from Africa.
Well, not wishing to brag as the temperature heads over 30 degrees again but the warm weather here in Gisborne made me think of the question we're often asked about the PCR reagents we sell from our European manufacturer Solis BioDyne. Yes, all their reagents (dNTPs, ladders, PCR masters etc) are stable at room temperature for up to a month and we're often asked 'how stable are they really?)
Solis regularly demonstrates the stability of their enzymes and the hot weather here made me think of a recent demonstration they showed - and kindly let me reproduce here. As they say, a picture speaks. . .
This article was originally published at BitesizeBio here
It’s the molecular biologist’s version of ‘I have good news. . and bad news’.
The good news is that I amplified the DNA band of interest. The bad news is that I amplified these other bands as well! Oh, and this smear. What to do?
Typically you might try and cut out the band of interest and gel-purify – an issue if it’s a lower intensity product and most of the DNA is lost in the subsequent purification. Or the products are more closely spaced than your dexterity allows.
Well, its probably the same as tempting fate when mentioning 'the Scottish play' . . .but then we're not overly theatrical so we can say MacBeth. However superstition may reign when you get the band on the additional lane on the gel or the extra qPCR amplification curve and think 'did I load template in that reaction?'. That's before the repeat reactions tell you that it's con . . .contam. . .the dreaded 'c'.
Thankfully instances of it are usually rare in real-time qPCR (hopefully only the one gel to analyse products when establishing an assay) but it can be a major hassle to eliminate.
One of the difficulties can be the research desire to know where the contamination is coming from versus immediately getting back to strong positives and clean negatives. In the former, often one reagent may be exchanged at a time - often in the vain attempt to avoid throwing out enzyme vials or master mix, while spreading the contamination further.
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 chemistries, your PCR primers still reign king in the reaction. Your probe design is not going to determine the efficiency and - usually - the sensitivity of your PCR and qPCR reactions. Therefore its worth paying a little care in looking after your PCR primers - especially if they're being used for a large number of runs and samples.
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.
This article was originally published at www.bitesizebio.com on 14/10/2009 here
One of the great features of PCR is its excellent sensitivity as we know. And many articles describe real-time qPCR as an added leap forward in that sensitivity – to the point where it has become a standard feature of a new assay description