Wednesday, March 28, 2007

Thou shall gel-purify PCR products!

Because otherwise you might just end up like me learning it the rough way. Admittedly, it involved the interplay of several «unfortunate» properties, but nevertheless it eventually made me streak 320 colonies and even worse cost a lot of time. You may wonder what my story is? Here it comes.

80 nicely streaked colonies It all started off with happily glaring PCR products bands implying that 99.9% of the DNA in my solution should be my product. The strategy planned an extra cloning step in very handy TOPO blunt vectors (Invitrogen). This is where my first fault occured: I decided not to gel-purify the PCR reaction due to the high product content. My second fault was to use ampicillin instead of kanamycin or zeocin plates as the manufacturer's instructions would have suggested [1]. Combined with an extremely high transformation efficiency (10 billion per µg DNA) of the Mach1 E.coli cells (Invitrogen) in use, this perfectly selected for the few ng of PCR template. To my defence; I didn't know at the time that my template was a mammalian expression vector carrying genes for ampicillin and neo/kanamycin resistance. A round of weird digest, however positively confirmed inserts by internal primers, and a couple of maxi-preps later I had a huge amount of template DNA, with - in my eyes - still very weird digest patterns [2]. The testing continued, extended to the restriction enzymes and their according buffers, and culminated in a freshly ordered batch of them. The results stayed unaltered though.

Redoing transformations on kanamycin strangely didn't provide any better results. In this case, I think Fortuna refused to guide my colony picking hand and I ended up mini-prepping template plasmids once more. After the flaw eventually became apparent, I played streaking-slave and had a lot of fun with roughly 320 pipette tips, the same amount of E.coli colonies, a grid, and 4 zeocin plates [see picture]. Plasmid DNA purified from the fittest cultures finally led to correct digest patterns. Oh, the joy of trouble shooting.

The morale of the story? Always gel-purify your PCR products! It saves you a lot of troubles. It's better to invest more time and money to do something completely right, than loosing time, opportunity, and money by having to redo something.

[1] This is the reason why one should always read that damn thing, or at least skim it.
[2] As I still believed I had my TOPO vector with the appropriate insert.

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Saturday, March 24, 2007

I make people think!

You can hardly believe it and I'm surprised myself that I'm not a major cause for headaches. But I've been awarded a thinking blogger award by Ψ*Ψ. Nevertheless, I'm not going to participate in tagging blogs and linking to the originator. Besides, I linked to my most frequently read pages anyway. Be asured that they all make me think; mostly that my chemistry knowledge is rather limited.


Friday, March 23, 2007

There's life in the old dog yet

My internet connection was recently as reliable as the London tube and therefore the lack of random entertainment from my side. I even bought a wireless PC card to upgrade my laptop to the state of the art of wireless computing. However, the nearby UCL access point is strangely still out of reach. Now it appears as if my halls IT manager [1] sorted out the connectivity problems.

Rather enjoyable were the recent articles in Nature and The Independent [2] about some British colleges awarding science degrees for pseudo-scientific subjects, such as homeopathy. A prominent figure in these discussions is David Colquhoun from the UCL Pharmacology department. The introductory paragraph from his website «DC's improbable science page» promises a couple of entertaining hours browsing it [3]. Enjoy!

This page is devoted to giving publicity to assorted dubious, erroneous, nutty, or downright fraudulent claims about drugs and other sorts of treatment. It includes, but is not restricted to, so-called Complementary and Alternative Medicine (acronym, SCAM). In particular, it is about the incursion of such ideas into universities.

Another interesting article about «Why do women remain curiously absent from ranks of academia?» was published in last week's Nature - on the last page [4].

[1] Or idiot, that's a synonym in this case.
[2] Scanned for your convenience.
[3] I'm especially found of the AIDS goat serum «cure».
[4] Pure coincidence?

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Sunday, March 11, 2007

Happy Birthday to someone special!

Another year has passed and Alex became a little wiser (espcially regarding stress in combination with being a medic). This song is only for you, my muse:

Miss you.


Friday, March 09, 2007

What does one do when the supervisor is in a meeting?

Given that digests are running and there's nothing else to do [1], the following:

Overture: playing with dry-ice.

Act I: The glove of unfailable polymerase chain reactions.

Act II: Mr & Mrs Glove.

Epilog: Facebook.

[1] Admittedly, that sounds overly keen, geeky and freaky. All that responsibilty talk must have influenced me more than I wanted (Mum, I don't wanna grow up).

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Wednesday, March 07, 2007

Party steaks

Persistently reading literature about the almighty and almost immortal [1] green fluorescent protein while simultaneously stumbling across the idea of making party jelly with fluorescein initiated another Mike-patented string of random thoughts. And in the end I saw it as clear as the back of my head: I'll have to breed transgenic GFP-cows (or pigs) to provide the ideal meat for black-light BBQ parties [2]. I already visualise a whole series of products available as GPF, YFP, CFP or dsRed variants.

In case I can't find a market in Europe, I'm quite sure the Japanese culture would be suitable to create a hype for my ingenious «invention».

[1]GFP resists temperatures up to 90°C and ignores proteases.
[2] Well, maybe not BBQ as the temperature shouldn't rise above 90°C, but the meat would certainly be suitable for poached dishes such as Tafelspitz. That would be a freaky Christmas dinner.

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Monday, March 05, 2007

Sodium source discovered in a biological laboratory! Discoverers aver to be innocent bystanders and gainsay any involvement in the debacle.

Anyway, the UCL Darwin Building has been subject to frequent lab-relocations as long as the complex exists [1] and the remainder of a more recent move was a shabby rusty old bottle of elemental sodium under paraffin oil. Of course my first intention was to throw the bugger off London Bridge and have a little fun with the gurgling hydrogen, but Andrea was strictly against any of my superb ideas. Eventually, someone from the chemistry department is going to pick it up and use it for more «useful» things [2].

[1] Already Darwin himself frequently moved his chest of drawers! Therefore, we merely stick to a very old tradition.
[2] I bet they wanna throw it off the bridge as well.


Thursday, March 01, 2007

My first meme ever

Only because po8 asked me to do so [1]. So I'll list 6 of my idiosyncrasies.

  1. As a toddler I worried my parents, because I refused to talk until I was two years old. However, when I eventually started I spoke in whole scentences (yeah, I'm a perfectionist).
  2. I never separate my clothes for laundry day. Dark, whites, coloured, all washed in one go.
  3. I'm absolutely anal about my books. If you borrow one and the back is buckled on return, I buckle you.
  4. Unlike po8, I need at least 8 hours of sleep or I'll get grumpy. I actually fell asleep in the world-cup final last summer, as I was doing an internship that forced me to start working at 7am.
  5. I learned crochet, knitting and sewing. One of those things you'll always hate your grandmother for (besides weeks of succini cream soup).
  6. I hate public toilets - call me Finch.

Your turn Ψ*Ψ or Excimer. And I guess propter doc wouldn't be disinclined to add another meme to her collection. Hepp, hepp, bugger off meme, fly away like a bird into the vastness of the blogosphere.

[1] In case you don't understand German you'll have to take my word for granted.

Addendum: I sometimes get caught cooking topless. The half-naked chef.