Eat, Sleep, Pipette, repeat

 

Setting up the next experiment. It involves pipetting agar growth media into (literally) 100’s of tubes – see movie clip above. I try to overfill the tubes with the molten agar mixture (this is important for a later step….where I place single Arabidopsis seeds on the agar surface…watch this space).

Science is 99% perspiration, and 1% inspiration….and this is the ‘perspiration’ bit.

 

 

“now acceptable for publication” :-)

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Ahhh….the best 4 words in scientific research? It’s been a long, arduous trip but finally we shall be adding a few¬†dents to our current knowledge of alternative splicing/splicing factors/temperature and the clock….will keep you posted.

Made me think about the time it takes to publish scientific research, and I came across this commentary article in Nature from 2016.

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I think many of us working at the coal-face of research will recognise a lot of what it says, e.g.

Many….feel trapped in a cycle of submission, rejection, review, re-review and re-re-review that seems to eat up months of their lives, interfere with job, grant and tenure applications and slow down the dissemination of results.”

Also talks about “resetting the clock” – not to do with circadian clocks, but related to the time stamp of submission and resubmission(s).

Is it taking longer to publish? One contributor to the article says that the average time for their group of papers took 9 months…[9 months is good, no?]

Anyway, for the time being lets focus on…”now acceptable for publication” ūüôā

 

Improved Gateway

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Trying to re-learn the language of GATEWAY cloning. ‘BP reactions’, ‘LR cloning’, ‘attB sites’, ‘entry clones’, ‘destination vectors’, ‘binary vectors’…I could go on. Thinking of trying a series of ‘Improved Gateway Binary Vectors (ImpGWBs)‘ to make plants express our genes of interest (GOIs) fused to markers. These are described in this paper:

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As for the choice of marker, there is a dizzying choice, so as well as trying to get to grips with GATEWAY, there is the question of what best to use as marker. I’ve had disappointing results fusing our GOIs to green fluorescent protein (GFP) in the past, so maybe time to try something else. As you can see, there is no shortage of choice:

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How long is a piece of stri…promoter?

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Trying to work out how long to make the upstream promoter region for some plasmid constructs. I’ve been using the SnapGene tool to visually stitch together DNA sequences.

How long is the promoter for a particular plant gene? 500, 1000, 2000 bp? or is it defined by particular features in the promoter? I guess for some genes the upstream region barges into other gene loci.

I tried out this web-tool, plantpromoterdb.

Seems quite useful. It displays some features of the promoter that might be considered when deciding the promoter chunk length e.g. it will show the predicted transcriptional start site, and other cis– features.

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Seems, though, that most studies take a nominal 1000 to 2000bp upstream of the translational start codon (ATG). Think it’s good to know some of the promoter ‘landscape’ though. Here’s the reference for plantpromoterdb:

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We are Stu-Dents

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Always amuses me the infographic from Matt Might¬†called ‘The illustrated guide to a Ph.D,’ (click here for the full infographic page). Very useful for new Ph.D. candidates, but it I guess it’s just as valid for anyone working at the coal-face of scientific research. We are all just trying to make small dents at the boundary of scientific knowledge – ‘suppose that makes us all eternal stu-dents!

 

Collecting Cobras 2

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As a follow-up to the earlier ‘Counting Cobras‘ post, Hugh pointed out this recent opinion article from The EMBO Journal written by Alain Prochiantz.

The author recommends playing the science ‘game’ – for example by trying to publish in the so-called high ranking journals even if they do not necessarily¬†reflect novelty and importance, because this is important for obtaining grant funding. Interestingly the idea of developing side projects appealed to me – the concept of planting seeds that, although would probably not get published in stellar journals or even accepted by peers short-term (see¬†‚ÄúYou should stop science‚ÄĚ, ‚Äúyou are making a fool of yourself”), but nonetheless with time might grow and branch out into research with high impact.

Other notable comments were the idea that “…there never really is a golden age [in science research]” and ” …it was not better yesterday but [..] it will be better tomorrow, provided that we never forget to defend the ‚ÄúValue of science‚ÄĚ.

and finally…”Science remains a game, a game that must be taken very¬†seriously, but nevertheless enjoyed”.

I suppose it’s trying to keep that balance – to play the game enjoyably!?

Coolit

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I’m liking the logo on my ice bucket today….what fun typefont is that?!

‘Polishing’ my RNA today – thats when we remove contaminating genomic DNA that might have co-purified during the¬†RNA isolation stage. I’m using TURBO DNase from Invitrogen (…or is it ThermoFisher?)

In this case RNA:good :-), DNA:bad ūüė¶

We don’t want DNA since it might interfere with measuring gene expression levels later on.

Collecting Cobras

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Interesting article last week – “performance-driven culture is ruining scientific research‘ – in the Guardian that has led to much discussion amongst the post-docs. All about the impact factor (IF) metrics – an arbitrary measure of how high (…or low) a journal is ranked. The article argues that a fixation on IFs, together with the Research Excellence Framework (REF) excise, detrimentally affects the diversity of Science research. The article starts off with an interesting anecdote about collecting cobra snakes during British rule in India…

Interesting also to see the range of comments after the article, ranging from – Science should be no different from other areas of work where performance led metrics are used ¬†– to there needs to be a change in culture, would Darwin’s or Einstein’s ideas have emerged in a performance led scientific culture? Oh, and someone else noted the cheesy stock photo (above) as a common sight in all labs!

 

Let there be light

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Often we need to measure light intensity for our experiments and we use this little gizmo to do the job – a LI-COR light meter. Measuring light intensity is quite complex, but I find this explanation from GroWell (Hydroponics & Lighting) to be very useful:

Photons are counted¬†in micromoles (¬Ķmol).¬†One ¬Ķmol is 602 quadrillion¬†photons! I can’t even imagine a quadrillion!

I took a measurement of outdoor light intensity in Glasgow in February – reasonably bright, fast moving clouds, occasional cloud cover, temperature around 4oC. As you can see there were around 160 x 602 quadrillion photons hitting the sensor per second.

When there was no cloud I got readings up to around 600 umol – light intensity really does rapidly change….moving to the shade of a building I recorded around 45umol

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When we grow our plants in environmentally controlled growth cabinet we set the light intensity to around 130-150 mol. I suppose this is equivalent to a reasonably cloudy day in Glasgow! Plants will be used to much higher levels of light intensity though….how do they adapt to such rapid shifts in light?

 

 

RNA day

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….a day for making lots of RNA. Using Qiagen’s Plant RNA kit with their lilac and pink tops.

A tad whiffy due to using beta-mercaptoethanol – hope my lab colleagues don’t mind too much.

Follow the Qiagen protocol pretty much as is, except for the bit where you add ethanol to the QIAshredder flow through.

Instead of adding 96% ethanol, we routinely add 70% ethanol – see point 4 in the picture below. This seems to result in a better yield and quality of RNA. Our samples are for Arabidopsis plants (quite old….around 5 weeks old) and sometimes these plants have experienced constant light so they have lots of polyphenols.

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