Transcription and Translation

Being “Good at the Bench”

I’m on the last bit of my protracted vacation. I’ll be back on Monday. To commemorate work (got to get back into that mind-set) here’s a little rant I posted last year.

Here’s a tale from the lab.

Today we had an interesting discussion. It started off with PBS and ended up on the topic of understanding the principles behind much of the protocols used in a lab.

It all started when a rotation student asked if there was a lab stock of PBS (phosphate buffered saline), a common buffer used in the lab. Another postdoc informed the student that there wasn’t a lab stock and she did not have any at the moment. The student had to either get some from another lab member or make the solution from scratch. Five minutes latter he showed up with a small packet of powder, one of these “add water, stir, and presto your solution is made” things. These packets are quite expensive in relation to the cost of making the solution from reagents off the shelf. I can understand that adjusting the pH manually is time consuming, however when I was a gradstudent (not so long ago) making these solutions wasn’t so hard. We had calculated the proper amount of dibasic to monbasic sodium phosphate, so that if you added the right ratio of the two components you would end up with PBS with the right pH. We also had TRIS tables (they’re at the end of the Molecular Clonning, a book we would simply call Maniatis after Tom Maniatis, the famed scientist and “senior author” of the book) to make TRIS buffers at various pH levels.

We then discussed another event. About two weeks ago, I was helping another student synthesize RNA in vitro. After purifying the newly made transcripts over a column, we had to exchange buffers. I told her to precipitate the RNA, “just like you would precipitate DNA” and resolubilize it in our preferred buffer. She then asked me whether she should precipitate it with TCA (trichloroacetic acid – a reagent that precipitates protein). I thought to myself, she had never precipitated DNA, could this be? And why should she? The only time she’s touched DNA is when she prepares it with a kit. Why would she know how to precipitate DNA?

So my point? I don’t want to rant about kits or premade, ready to use PBS packs (I’ve already commented on kits and subsequently got attacked by a couple of kit lovers). My point is that kits are black boxes. The more we scientists use these kits, the less we understand how basic lab techniques actually work. And the less we know how lab techniques work, the less we are able to trouble shoot. Now kits do have their advantages. Although homemade preparations often yield better results, kits are more reproduceable and reliable. I remember on many occasions when the pH of a single solution was off and all of my DNA preps failed, now that never happens. But if we want to get mRNA into cells, knowing the intricacies of RNA precipitations including which salts to include and what buffers to resuspend the RNAs in can make a huge differences.

Here’s another example. Some individuals in the department have tried on several occasions to microinject tissue culture nuclei. They purchase prepulled microinjection needles and use automated microinjectors .. but in the end just can’t do it. I pull my own needles and write my own programs for our needle puller, I constructed my own microinjection setup (with no electronics components) I even use a syringe instead of a pico-spritzer that automates the pressure flow into the injection needle. Consequently I am able to microinject nuclei reliably month after month. The difference is the dept of understanding. I can adjust my needles, regulate pressure and adjust many parameters of the microinjection procedure. Those that automate the whole process simply don’t have the same capacity to adapt. Why do you need to adapt? Well not every cell type is the same. Different microinjected substances require different needle etc. I also have plenty of little tricks. It’s all this knowledge that allows me to reliably perform this technique. Am I just good at it? Not really. I’ve already trained three others and they are much more capable of microinjecting than the other people who used the automated setup.

My point is that it is fine to use kits or automated microinjectors or premade PBS packs … yes, it’s fine to take the easy way, but try to at least understand how your kits work. Such information can help you troubleshoot, or adapt, and in the end that is the difference between average and “being good at the bench”.

Comments

  1. #1 Bill
    October 12, 2007

    trichloroacetic acid – a reagent that precipitates protein

    TCA also precipitates nucleic acids. I wasn’t sure about RNA, which is why I looked that protocol up — but I’ve been doing thymidine incorporation assays, in which TCA is used to precipitate DNA and allow removal of unincorporated labelled thymidine.

  2. #2 Oliver
    October 14, 2007

    For minipreps I use home-made alkaline lysis most times, and a kit everytime I need clean stuff for sequencing or just high quality DNA.. so I tend to compromise. I can’t believe how many people don’t know how to make chemically competent cells by themselves, and buy them although they use them often. It takes basically no time at all and is much cheaper!! I do think there’s a big difference in the level of understanding of “kit” scientists, and those that are comfortable with both.. In the end though I think it really comes down to who trained you.

  3. #3 Joolya
    October 15, 2007

    Kits are great for some things; e.g Roche’s High Pure Maxiprep kit with the columns means maybe I get a little less DNA, BUT I never lose the pellet during the final precipitation step. On the other hand, for some things I want to be able to adjust single parameters, which kits don’t allow. I like to know which steps are critical and which steps are expendable … you know, sometimes “5 minutes” means really, seriously, 5 minutes and other times it means “enough time to go to the bathroom or check email once”. Or, sometimes pH 7.0 means really, seriously, pH between 6.9 and 7.1 and other times it means anywhere from 6-8. This I like to know for both scietific troubleshooting reasons and also for reasons of laziness.
    Finally, (my six cents) a lot of cell biology is voodoo magic (sorry, but it is) which requires getting a feel for. Live cell microinjection is one of these things. Maybe a robot could do it more reproducibly but it would not be as successful as a human agent with a lot of practice and good hands. When I used to do a procedure similar to microinjecting (lifting single cells from culture by squirting them with trypsin and sucking them up) I liked to 1) pull my own needles and select the best needles for the job and 2) pipet by hand, not with the microinjector which I felt was too bulky. The downside was that the procedure was never exactly the same day to day – the upside was that it was almost always successful!

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