As described previously

Greetings Earthlings!

For this blog post, I urge you to close the PhD Comics/xkcd window that you have open and open http://www.nature.com. (Dude, if you don't know what comes up if you type this in the address bar, make sure your advisor doesn't know about it. (S)He'll have a fit.) There's a "Search" tool on the top right side of the page.

Click¹ on the "Advanced search" option and type in the search term "as previously described" and from the drop-down menu² menu, select "The exact phrase" and click¹ on "Submit". If you have followed the instructions to a tee, this is what you should see:

->24,158 results for "as described previously"

Now do the same for "as previously described" as described previously².

->22,531 results for "as previously described"

What this probably means is that there are a lot of half-sane people out there who are desperately looking for a protocol, but are not finding it where it should be! Why Nature even allows something like this is beyond me.

Until journals take a stand against incomplete and ambiguous protocols, be assured that you are going to play a never ending game of Hide (by the protocol writers) and Seek (which you will try with little luck).

Until next time,

Visual Acuity.

¹ Take the cursor to it and press down down hard on the mouse at the place where your index finger is.

² That thingy that has an arrow pointing down. When you click¹ on it, it magically gives you a list of options.

³See how infuriating this is??

It's just human Nature

Greetings Earthlings!

In this post, we're taking it up a notch with a review of a protocol from a paper published in nothing less than the journal giant -- Nature! So put your hands together (not in applause, but in prayer; we're hoping to come out of this venture with a sound mind) for Ishiguro et al.'s 'Shugoshin–PP2A counteracts casein-kinase-1-dependent cleavage of Rec8 by separase' from Nature Cell Biology's April 2010 edition.

I'm sure it's a great paper, but let's quickly move to our section of interest -- "Methods". The Merriam-Webster online dictionary describes 'method' as "a systematic procedure, technique, or mode of inquiry employed by or proper to a particular discipline or art". Let's see if that definition holds true.

Take a look at the 'Chromatin Immunoprecipitation assay' (I have this sudden urge to binge on  potato chips and hence the ChIP protocol. All right, I agree that was a bad one.)


Chromatin immunoprecipitation assay.
1. Anti-rabbit IgG, anti-Rec8 [ref. 26] and anti-GFP (Living Colors Full-length A.v. Polyclonal Antibody; Clontech) polyclonal antibodies were used for immunoprecipitation.
2. DNA prepared from whole cell extracts or immunoprecipitated fractions was analysed by quantitative PCR with the ABI PRISM7000 system (Applied Biosystems) with SYBR Premix Ex Taq (Perfect Real Time) (TaKaRa).
3. The primers used for PCR were described previously [ref.25] except zfs1´ (5´-CAGTATTGCTGTACAAAGATGTTA-3´ (OLI559) and 5´-ATTTGCTAACTAGCCCAAAACGTC-3´ (OLI560)), 7E8 (5´-TGTCAACGTTGACAAGGAATCATT- 3´ (OLI561) and 5´-CCACGGAGTCAGCTACATCAATGT- 3´ (OLI562)), 1105(5´-GGTTTTGCCGATAACTTCTGTAAT-´ (OLI563) and 5´-CACCATTGGGAATTGCCAGCTTCA- 3´ (OLI564)) and abp1 (5´-CGTCCCCTGAGCGGAATTATTTTT-3´ (OLI565) and 5´- ATAAACTGGATTACCAGTAAGAGT- 3´ (OLI566)).
4. Immunoprecipitation by anti-rabbit IgG or by anti-GFP antibody in the untagged strain was accounted for as nonspecific binding in chromatin immunoprecipitation fractions of anti-Rec8 or anti-GFP, respectively.


FYI no one, I repeat, no one ever publishes protocols in papers with bullet points! This assay has been split up into readable chunks by yours truly for the sake of this blog post (Hey that's the anagram of blogspot! Please excuse my random rantings). That's another question for the protocol writers : isn't it hard enough  already to understand protocols? Why make them harder by writing them in the form of paragraphs?

Anyway, coming back to where we were, if you take a look at this protocol, you''ll see that there is none. In fact, it would be appropriate to rename it as Outline of

What-we-did-in-the-lab-to-get-the-results-we-got-and-aren't-in-a-million-years-letting-you-know-unless-you-are-prepared-to-grovel-and-ask. Let's attack the problem step-by-step.

Step 1 tells us what antibodies were used for immunoprecipitation. Great!

Step 2 makes DNA extraction and PCR seem like non-entities. Question time!

- How was the DNA extracted?

- What were the PCR conditions?

Step 3 again tells us how the primers used for the PCR (mentioned) in step 2 are the same as those used in a different paper's methods, with the exception of (the primers listed in the assay..). Incidentally, this step takes up the most space.

Step 4 tells us how to interpret "immunoprecipitation in untagged strain".

I just have one more question : how *does* one carry out this "Chromatin Immunoprecipitation assay"? I'm sure a lot of biologists have "Oh! The 'Methods' section is the least important of all the sections in the paper" or "It takes up too much space/time to write complete steps in a paper" or "Shania Twain is my favourite singer" as a response to this question.(OK probably not the last one)

For novices/robots all these reasons are immaterial. If you try to automate this protocol, you sure are going to have one confused robot.

Judgement time! So how many QM's does this protocol get? Being a democratic blog (except for the fact that I am the sole deciding factor in what goes into the blog), we will let the people decide again.

Until next time,

VisualAcuity