I was perusing the feeds of my fellow ScienceBloggers the other night when I came across a post by ERV that really resonated with me. In it, she expounds on the benefits of doing things "old school" in the lab, specifically with respect to having hard evidence to defend oneself if ever accused of scientific misconduct. She has a point, but that's not why the post caught my attention. I've actually been struggling with the conflict between "old school" and "new school" recently.
You see, I've recently been in the position where I've had to add people to my lab, and in fact the entire staff of my lab has turned over completely in a brief period of time. Such transitions occur from time to time, but this is the first time one this radical has happened in my lab, as I've only been a principal investigator since late 1999. The new crew is very different from the old crew in a number of ways. For one thing, their knowledge and talent distribution is very different. My old crew was hard core molecular biology. I'm talking cloning a complete human gene from ten introns, linking them together to form what we liked to cal a "Frankenmolecule" cDNA and liking it; bashing the promoter of that gene and then doing chomatin precipitation assays to cover 20 kb or until we found a regulatory element (whichever came first); measuring the copy number of mRNAs in a single cell; identifying microRNAs; doing confocal microscopy on four different fluorescent probes and then confirming it with multiple co-immunoprecipitation to confirm protein interactions of any colocalications observed hard core. Don't worry if you don't understand all that. Those who do understand will know just how hard core (and to some extent old school) I mean, and for the rest of you suffice it to say that I mean really hard core--at least for a surgeon's lab. (I'm not DrugMonkey or PhysioProf, after all, but I do pretty darned good for a surgeon, if I do say so myself.) The new crew is more cell biology-oriented and has shown what to me are some surprising blind spots when it comes to basic molecular biology. I'm talking not even knowing how to do a good transfection. I realize now that in trying to put together a good new team with different skills that I may have gone too far towards the cell biology end. Fortunately, they're all pretty smart and should be able to pick up the techniques that need to be picked up. Their new skills will also allow me to do stuff that I couldn't do before (particularly with regard to vascular biology and studies with pericytes and models of inflammation, for which I didn't have the expertise before), but I find myself explaining a lot of things that I haven't had to explain for years. I think I'll be able to whip them into shape (being able to learn is more important than any specified knowledge base), although none of them will ever match my last post doc for his mad skills at molecular biology and there has been (and will be) some frustration on both sides along the way.
One thing, however, where I have run into intermittent conflict, however, and it has nothing to do with differing skill sets. It started when my new crew bought pre-cast polyacrylamide gels. Older faculty will understand why this may have precipitated my first disagreement with the new crew, as may some graduate students.
First, I'll try to stop slipping into jargon. Polyacrylamide gels are used to separate proteins in a procedure known as a Western blot. In a Western blot, also known as an immunoblot, is a method used to detect specific proteins using antibodies. First, protein extracts are made from the cells or tissue of interest, and the proteins are denatured (unfolded) by chemical means with strong reducing agents that break a type of bond called disulfide bonds. This eliminates the secondary and tertiary structure, allowing them to be separated purely on the basis of molecular weight when forced to migrate through the gel by placing the gel in an electric field. After the proteins are separated, the gel is then placed on a special membrane, and again using the application of an electrical field the proteins are transferred to the membrane, where they are immobilized. They're then "probed" by incubation in a solution of an antibody against the protein of interest (the "primary" antibody), which is detected by means of a "secondary" antibody directed at the species-specific part of the primary antibody. It's one of the most commonly used techniques in any lab that does molecular or cell biology.
And, like ERV's boss, I guess I'm fairly old school (although not nearly so old school that I insist on CsCl gradient purification of DNA). Biomedical scientists out there will understand what I mean when I say that I'm getting a radiation license because I still think that 32P labeling produces better results when doing gel shifts. I'm so old school that I still like Northern blots.
Now here's where my old school mentality came into conflict with the new school. Actually making the polyacrylamide gel is a bit nasty, as explained in detail here. Why are they so nasty? In brief, you have take two glass plates, clamp and tape them so that they form a thin space between them into which to poor the liquid that will become the gel. Then you have to mix the correct proportion of acrylamide and bis-acrylamide, add a chemical called ammonia persulfate, and quickly poor the mix into the space between the plates (i.e. "cast" the gel). As you might expect, leaks are not uncommon, and, given how close together the plates are, bubbles can be a maddening annoyance. Sometimes for no apparent reason the gel won't polymerize at all, necessitating recasting it. Even better, unpolymerized acrylamide is a neurotoxin. Still, casting a good polyacrylamide gel and running a high quality Western represent what I thought to be indispensable skills for any biomedical scientist. Moreover, there have been many advances since I was a graduate student to make casting these gels easier, including clamps and holders that make the possibility of a leak much less compared to the old school way of putting two spacers between two specially cut pieces of glass, taping them together, and hoping like hell it didn't leak when you poured the acrylamide in and that you didn't get any bubbles that tapping the plates wouldn't dislodge. It never occurred to me in my entire career since I poured my first polyacrylamide gel during a student rotation some 25 years ago not to pour my own gels or that anyone else wouldn't pour their own gels either.
They do, though. Now you can buy precast gels. All you have to do is take them out of the plastic package take a piece of tape from the bottom of them, mount them in the gel apparatus, and you're good to go. Some even have molecular weight markers built in, making measuring the size of the separated and detected proteins a snap.
So what's the problem? For one thing, they're way more expensive than pouring your own. (It just occurred to me that that sounded like "rolling your own," but I assure you it's not the same thing.) Of course, this is just the raw materials. Pouring a typical gel can take a half an hour or more before samples can be loaded and the separation begun. If you factor in a technician or postdoc's paid time such precast gels may be cost effective. Indeed, that's the argument made for all manner of kits in "new school" molecular biology. You can now buy "master mixes" for PCR that have all the ingredients, including magnesium, nucleotides, salt, buffer, and the enzyme that does the magic that is PCR, Taq polymerase, in them already mixed up. Around the time that I was finishing graduate school, suddenly there were Qiagen kits that make isolating plasmid DNA far easier and less messy than the old-fashioned mini and maxi preps that I used to do as a graduate student. At that time they were so expensive that my thesis advisor kept them in his office, and we had to ask him for them when wanted to use them. (Actually, I love Qiagen kits; they're so much easier and produce cleaner DNA than mini or maxi preps, albeit at a price of a lower yield.) Buffers are all pre-made and ready to go, no muss, no fuss. There are now kits that let you isolate RNA from cells and tissue without all that messy mucking around with toxic guanidine thiocyanate, phenol, and chloroform. Everything is reduced to an easy-to-follow recipe using kits. Add enzyme A to solution B (don't worry, you don't need to know what's in solution B; it's proprietary anyway). No more film for detecting the chemiluminescence used to detect proteins in Western blot; we use computer imaging instead. Ditto the detection of radiation from 32P used to label DNA.
You're just being an old fart, youngsters in the lab might say to me. Maybe so, but I don't think that entirely explains my misgivings about the proliferation of these kits. Something is lost when a scientist relies too heavily on kits, no matter how convenient they can be. I have a new technician in my lab who had done some graduate school. She's smart and hard working, but she'd never cast a gel herself except for once under supervision in a class. Otherwise, she used precast gel. She has no clue how to troubleshoot it if a gel doesn't polymerize or if it doesn't run right, producing smears instead of nice, tight bands. She's never had to. Yet in never having learned how to cast a gel, I suspect that she doesn't fully understand just how the gel works to separate proteins. She doesn't have that intuitive, visceral understanding gained by fiddling with the components needed to make a polyacrylamide gel and troubleshooting when things go wrong. In science, one of the most important abilities is to be able to differentiate when an experiment isn't working because the science doesn't support your hypothesis and when an experiment doesn't work because of a technical problem or because you're screwing it up. Kits supposedly make the latter possibility much less likely, but over-reliance on them makes it far more less likely that you'll be able to identify and fix the problem when it is the latter of the two.
Pre-cast gels are actually a relatively minor thing, though, when it comes to my concerns. If I do lab work again (which I do from time to time), I'd probably use them myself because an hour of my time casting the gel is costing my grant and my university far more than any precast gel costs. In any case, I've heard the other arguments in favor of kits as well, perhaps the most common (and compelling) of which is that they make basic techniques easier, freeing researchers to think more about the overall design of their experiments or to get more experiments done in the same amount of time. These arguments also postulate that a theoretical understanding of how a technique works is all that's necessary. Maybe so, but I don't always find that argument compelling. For one thing, one's understanding of the science behind a technique can only be strengthened if one has some practical, physical exposure to the technique and how it is done. For another thing, as I mentioned above, what happens when the kit doesn't work? For example, what happens if your RNA yields are crappy using a Qiagen column? How do you troubleshoot? Sure, you can read the troubleshooting section of the manual, but what if the problem is something that happened to your sample before you use the kit? If you've never seen it done any other way than by a cookbook-like following of a printed protocol written by the manufacturer of a proprietary kit, you'll probably have a hard time figuring it out. I've seen students and even postdocs who've had no clue, for instance, how to optimize conditions for PCR other than by fiddling with the temperature cycle. It never occurs to them that the concentration of magnesium can have an enormous effect on the efficiency and specificity of the reaction. They've always used a kit with a set concentration of magnesium, fiddled with the temperature cycle, and it usually just worked. When faced with a reaction that's producing multiple products (seen as multiple bands), they have no idea that lowering the magnesium concentration can help with that every bit as much as increasing the annealing temperature.
Old fart or not, "old school" or not, I'm not totally opposed to kits or "recipe" science. I do, however, have definite ideas about what its role should be. That role should not be for beginners who are just learning science and laboratory technique, at least not as exclusive means of doing lab techniques. Old school techniques are an important learning experience and can do much to help new scientists develop an intuitive understanding of the science behind them, as well as valuable troubleshooting skills. At this stage of a scientists' career kits are little more than crutches, and at some point the crutch has to be thrown away. However, once a scientist has reached a certain level of proficiency, he or she already has developed that understanding, and kits then become very valuable as a convenient time-saver. Moreover, more senior trainees and postdocs have higher salaries, and if there's one thing I've learned as a PI it's that time is often more valuable than money.
In the end, I've come to the conclusion that it more or less depends on the kit, as some kits are more useful than others, namely ones that produce a better end result. That's the reason I generally like Qiagen columns and kits. They generally in my experience produce cleaner, higher quality DNA preps than mini preps and maxi preps, and they're certainly a lot less nasty for isolating RNA than using the old school guanidinium thiocyanate/phenol/chloroform method that I "grew up" with as a scientist and don't require all sorts of toxic chemicals. On the other hand, I'm less enamored of precast gels, which strike me as a long run for a short slide and are expensive to boot. And I really don't like "master mixes," where I don't control the concentration of every reagent. The bottom line is that I probably lean more towards "old school" when it comes to molecular biology kits, but I do make a fair number of exceptions for particularly useful kits that clearly produce better results than the old way.
This is similar to not knowing math inside and out because you've always used the calculator.
for PCR that have all the ingredients, including magnesium, nucleotides, salt, buffer, and the enzyme that does the magic that is PCR, Taq polymerase, in them already mixed up.
I first read that as butter (probably because it followed salt).
I teach AP Bio and we do some basic electrophoresis activities as part of the lab component. I do have them pour their own agarose gels - I think it's an important lab skill for them to have before heading off to college. Now, agarose is a lot more user-friendly than polyacrylamide, but it still takes a long time to teach them how to do it - and about half of them wind up with unusable gels due to ripping as they remove them from the mold, etc. It usually takes 2 class periods to get enough gels to use for an electrophoresis lab which takes 1 class period. So we compromise - they pour the gels for the first lab, and I pre-pour them for the second and third labs. It's the best we can do given time and budget constraints - but I still don't know if it will actually help them in college.
It has been a long time since my grad school days, and I've long since left the sciences for my illustration career, but-- OMG PCR MASTER MIXES, SQUEEEEEEE!!!!!!! (Cue the Monty Python sketch, "Back in my day, we used to DREAM o' livin' in a cardboard box!")
As one old fart to another (albeit in different fields), I can only suggest two things:
- I beat lab basics into my kids (both biological and organizational) with the lessons learned from not appreciating them when I was a student and then finding out in The Real World how much can ride on them (read $millions$ from just one example.)
- I offer them a deal: they are free to use the shortcuts as long as the work checks and things run smoothly. As soon as they have a problem that they can't explain precisely and verify then it's back to basics. Step by step, the hard way -- because, as we both know, there really is no substitute for having a "feel" for the whole process.
So far, seems to be working. The bio-kids even thanked me on Father's Day for, of all things, teaching them proper lab practices.
You forget to mention one benefit of pre-cast gels--consistency. You can be pretty much assured that the gel you bought in 2004 is the same as the gel that you bought yesterday and will be exactly the same as the gel you buy in 2014.
When you have different students moving through your lab, each with sometimes wildly different skills and experience regarding gels, it's nice to know that variability from the gels is being eliminated.
I love using kits, which save me a lot of time, but I'm also very grateful that I had to learn the principles behind each step when I took my core courses in graduate school. The first lab I worked in had a PI who insisted on our doing everything old-school at least once. I think he took that approach too far on more than one occasion, but I'm also happy that whenever I use lysis buffers, precipitation solutions, cleanup columns, and the usual menagerie of enzymes, I have a good understanding of what's actually going on in my Epi tubes.
One thing that I think really should be hammered into the heads of new lab workers is that they have to plan out their procedures. Whether you're using a kit, bulk reagents, or homemade solutions, it's a real problem when you have a few hundred samples laid out on your bench and then realize that you either (a) have started a two-hour job but have to leave for another commitment in forty-five minutes, or (b) have taken perishable material halfway through a protocol but have run out of a reagent that the stockroom can't obtain on less than 48 hours' notice. I've seen things like this happen to the same people over and over, and then they wonder why they get so frustrated at the bench. Proper lab practice includes budgeting your time so that you can actually get the work done with a minimum of screaming.
OMG! You're using a kit!!!
My thesis advisor used to admonish us that we hadn't purified our own restriction enzymes so we didn't *really* understand how to do a restriction digest. Indeed, her thesis advisor complained that she never had to incorporate 32P into ATP before making labelled oligos.
Give me a big fat break. There's a reason that biology moves faster now than it did 10 years ago - it's because we don't make our own oligos anymore (yep, there used to be a lab down the hall that supplied all of our oligos - that was how the guy got on peoples' papers). We don't run our own sequencing gels anymore. And we don't do CsCl DNA preps. (Yes, I know how to do all of those things, and I'm glad I don't have to anymore).
Nothing is lost by this. What we gain is that we are able to do science MUCH faster, and as a result, nobody gets a thesis for cloning and sequencing a gene anymore.
Every generation has their old farts complaining "graduate students don't suffer anymore, they buy PRE-CAST GELS!". But grad students still slog away, they just get more done than the grad students of yesteryear.
1) On the composition of your lab-
Don't worry too much about having great cell biologists- using FRET instead of co-IP is not the end of the world. Appreciate them for the expertise they have, while getting them up to speed on the necessary mol bio stuff.
2) On lab management-
I'm sure you already know this (since you sound like a decent fellow), but it's probably not a great idea to express your amazement at how much they don't know. And, of course, things will now take much longer than when Fomer Post Doc w/Mad Mol Bio Skillz (tm) was doing it.
3) On gels-
"In brief, you have take two glass plates, clamp and tape them..." (emphasis added) WHOA, Old Man, tape?! That is old skool. I hope we can all agree that there is relatively little to be gained by troubleshooting TAPE as opposed to using proper gel casting assemblies that facilitate good seals?
One thing you omitted about pre-cast gels is the virtues of gradient gels. Gradient gels work better (especially for some proteins) and I've still not figured out if it's even possible to do it by hand (I've certainly never seen anyone do it).
In my current lab, I do pour my own gels for most purposes. I've yet to have one fail to polymerize- so I've never "troubleshooted" them. I follow my recipe (thank you Sambrook!) and it works. I've never gotten this "intuitive, visceral understanding" of gels you speak of.
4) On the risks of kits-
I will grant you that more pre-made supplies and kits can be dangerous- but I don't think it's because you loose some mysterious cosmic comprehension you can only get if you have to fiddle with more parameters. If you get poor quality bands, it's easy to check your denaturing conditions, sample buffer, and batch of precast gels- you just have to remember that any of them could be a problem. Assuming the infallibility of stuff that comes from a company is a huge problem. Of course, assuming anything is correct can be a huge problem (I will spare you the horrifying story of the mislabeled primers). The key scientific skill to combat this, as I see it, is being able to come up with and interpert procedural controls.
Suffering (be it in time, headaches, or exposure to dangerous substances) will never be in short supply in a research lab; don't fetishize troubles (even when they lead to educational troubleshooting). Your trainees may not have to pull their hair out wondering why their gel didn't polymerize, but they will still wonder why their positive control FRET signal didn't light up.
Bah, real scientists blow their own glassware.
Becca - I wondered about the gradient gel thing, but I barely ever do Westerns, so I didn't have the guts to bring it up.
To the post - I do barely any molecular biology (being an embryologist), but I pride myself in knowing what every step in any kit or procedure I do is for, and usually a brief understanding of what's in it. Yeah, I'm one of those geeks who read's the front of Qiagen kit handbooks because I'm curious.
Also, as an only occaisional Western User (haven't broken single figures in the last two years), it's extremely helpful to not have to learn how to make gels from scratch for something I rarely do. I'm sure if you do ten a day it becomes as easy as falling off a bicycle.
The real divider between "old school" and "new school" in our lab is bacterial manipulation - do you use flame-cleaned metal innoculation rings and spreaders, or do you use disposable plastic spreaders and P200 tips?
Medicine isn't the only place it's happening, my friend.
My father is a long-time mechanical engineer. Recently he participated in a project where industry professionals partner up with engineering students on design projects for real products. He expressed frustration to me at how the students he worked with, though very smart, were unable to machine even the most basic parts. Time spent at a machine was, for him, a big part of an engineer's training -- knowing what it is to make a part to specification -- and today's kids are, apparently, not getting enough of that. I think across the board the nature of training in scientific jobs is changing from creating professionals to creating drones. Why? I don't know. More profitable? Increase the talent pool (meaning scientists/engineers are now more disposable)? All I know is it's kind of depressing.
Don't read during lunch. When I worked in genetics labs ten years ago, 'old school' meant pipetting everything with your mouth-E. coli, mouse brain puree, vinegar worms, toxic reagents-you name the putrid substance and my coworkers would mouth pipette it. Makes me feel a bit sick just remembering it.
@ Becca & Confused:
Gradient gels by hand are actually quite easy!
Make two gel solutions, e.g. 6% & 20% acrylamide. Add APS & TEMED to each. If your gel volume is 10 mL, draw 5 mL of 6% solution, followed by 5 mL of 20% solution into a 10 mL pipet. Density differences will keep them from mixing. Now carefully draw some bubbles into the pipet. As the bubbles float up, turbulence will mix the two solutions near the interface.
It helps to add a little bit of bromophenol blue to the 20% solution. Then you can get a visual estimation of how much the two solutions have mixed. Keep drawing in bubbles til you're satisfied, then pour your gel. If needed, you can add extra 6% solution to the top of the gel to completely fill it. Obviously, you need to work quickly enough that it doesn't polymerize in the pipet.
I've done this a couple of times, and it works reasonably well. You might expect that gradient variations across the width of the gel would cause wavy bands or large mobility differences between lanes, but that doesn't seem to be a big problem. At least, not in my limited experience with this technique. Obviously, it's not as good as using a gradient maker (or better yet, buying precast!), but it's adequate for some applications (e.g. if the linearity of the gradient and the reproducibility between gels aren't critical). Not sure where I learned it - probably one of those tips in Biotechniques.
P.S. I'm one of those old farts who learned to pour gels by hand in grad school. Using the magic yellow 3M tape. And these were 150 cm tall sequencing gels. (Maxam & Gilbert; none of that sissy Sanger sequencing!) There was a whole art to the taping process. I don't miss any of it. I'm fine with or without precast gels, but don't ever try to take away my Mini-Protean II!
For those who think science should be treated as some kind of industrial substance that emerges from a lab like goop from a "Play-Do Fun Factory" and is dumped into scientific papers, sure, there's not reason to ever move away from kits. Yes, they can be faster (as long as nothing goes wrong) and more consistent.
However, since we seem to be talking about students doing the science: we/they are not there to "ship product". We're there to learn science. Doing that properly requires not merely "knowing" but understanding why the science works - and in my experience that really does require a lot of do-it-yourself experience.
"you don't need to know what's in solution B; it's proprietary anyway"
That drives me absolutely stark screaming buggo as a student. Why does this reaction work? What does that tell me about the properties of my sample material, which knowledge I may be able to usefully apply elsewhere? Sorry, we're not allowed to know. That Secret Proprietary Special knowledge. Just use the kit and shut up, kid.
I imagine as robotics advances, we'll be seeing "surgery kits" sometime in the next couple of decades. I think Orac will agree with me when I say I sure hope if I ever need, say, an emergency appendectomy that the surgeon has actually spent some time in school learning to safely cut people up, rather than spending all his time merely getting "vocational training" on the AppendiMax SurgiPro 3000 Emergency Appendectomy Kit. Especially if it turns out my appendix is JUST outside the normal location for an average appendix...
"Bah, real scientists blow their own glassware."
You know, I'd actually LOVE to find somewhere to learn this skill. It seems to be a dying art (at least in the US). Plus, it'd make my brewlab look really cool.
Does anybody even teach this anymore?
I have to admit that as a young pup (in academic years), that this post smacks of "walking up-hill both ways". But here is my suggestion for having your cake and eating it too:
Use the pre-cast gels as a reward. Once you've published your first paper making all your own gels, reagents, etc..., then you can use pre-cast gels. Hell, why not take it a step further? Have a lab contest to see who can do successful PCR using only a few hot plates and some pots and pans. You could call it the "When I Was Your Age" contest. The prize will be a bag a black jelly beans ("we didn't have all those fancy flavors when I was your age").
But seriously, pre-made stuff is great for people who could have made their own stuff. But chances are that if you don't have the skills to pour a decent a acrylamide gel, using a pre-cast gel isn't going to save much time because you'll just mess something else up anyway. And that is why I don't work in the wet-lab anymore.
1.5 meter sequencing gels?!
1. The "proprietary" information is sometimes in the patent, which is publicly available.
2. I have never seen a kit that does something in some special, completely new way. It's always based on the tried and true "dirty" lab methods. I was having a problem with the buffer in a T7 transcription kit. I made my own buffer from scratch. It worked. Even though the buffer composition was "proprietary" and not publicly available. So if you don't know how a kit works, you generally just don't care. Which is fine - you use about a thousand tools a day, and you don't need to know exactly how each and every one of them works.
3. Everybody uses tools that they don't completely understand. I don't know anybody who still knows enough organic chemistry to describe how each and every reaction that you perform daily works. It's not necessary, although it's potentially useful. Like pouring your own gels. Maybe if we had infinite time, but most people in biology like to focus on the biology more than the procedural chemistry.
You would use a gradient maker to pour a gradient gel. It has two chambers connected at the bottom that allow the solutions to slowly mix, with tubing for pouring the gel connected to the chamber holding the heavier solution. The fact that Becca does not know (no slight to her) that you can make such a thing "by hand" illustrates the old school/new school divide. My issue with pouring gels is that it takes a long time, but my experience is dominated by the older apparatuses that take much longer than new ones. What is galling is that you will still get complaints about pouring gels with an apparatus now being burdensome to the new kids, when the real difference in time is much less than it used to be.
What kits allow one to do is to do harder and more complicated experiments in less time, or have less scientific experience or knowledge and accomplish similar amounts of work. Labs are becoming data production facilities where trouble shooting skills and deep experimental understanding, or technical facility over a breadth of areas are becoming less common. The demands for just having hands to produce data are driving the trend of less individual skill in some labs. Science is trending toward larger and larger teams as opposed to individual researchers being responsible for their entire projects. This allows workers that don't necessarily understand the fine details that underpin the techniques they are performing to do just fine. What used to be variables in the experiment that needed to be understood have just been turned into "some assembly required."
Or at least the above is part of the "new/old school" argument/divide.
Sorry - should have been 50 cm. I blame the rhinovirus that's currently propagating in my upper respiratory tract.
Yes, I've used gradient makers too. My point was one can make a serviceable gradient gel by hand, without even a gradient maker.
As for precast versus hand-poured gels, I'm all for precast if the expense is acceptable (as it often is). OTOH, it sure is good to know how to hand-pour for those times you run out of pre-cast and don't want to wait for a new shipment.
If you have students complaining how complicated today's apparatus is, I suggest letting them try 3M tape and some binder clips. That should shut them up fairly quickly.
I have total street cred! I poured thousands of 0.2mm thick radioactive sequencing gels as a grad student!! w00t!!!!
They've always used a kit with a set concentration of magnesium, fiddled with the temperature cycle, and it usually just worked. When faced with a reaction that's producing multiple products (seen as multiple bands), they have no idea that lowering the magnesium concentration can help with that every bit as much as increasing the annealing temperature.
Do they know that spiking PCRs with a bit of DMSO sometimes works a charm, either to fix the no-band or multiple-bogus-bands failure modes?
I love the fact that the magic ingredient in the "special" PCR buffer is almost always DMSO, or some related chemical.
PP- I want a real estimate on the number of sequencing gels you've poured, because it is the one thing that I really think I have done more of in my life than any other lab thing.
I know I have literally poured thousands of those things. I buy S2 rigs off of eBay!
I prefer a mix of kit-based and old school methods in the lab, but can switch to entirely old school when funding is tight (as it is now). Much of what I do (microdissections and primary neuronal cultures) can't be "kit-ified", but I'm glad there are things like Qiagen RNeasy kits to simplify processing all those tiny bits that I spent hours microdissecting.
I prefer pouring my own gels for Western blots, mainly because I like to tweak the polyacrylamide concentrations to optimize band separation for proteins of interest ... usually I'm comparing expression across multiple tumor cell lines, or different treatments for a given tumor cell line, and consistency is critical. The precast gels are limited as to acrylamide concentration and well configuration. With the mini-gel systems, it isn't that oppressive to pour 4-6 gels in the morning, and there are some very simple tricks to make your PAGExperience more pleasant.
Got leaks? Take a bit of the gel mix out (pre-TEMED addition) and add excess TEMED to this in a small beaker...distribute across the bottom of each set of plates in your casting frame (about half a centimeter of this "fast-cast" solution will do the trick), and when it polymerizes (quickly!), it will seal the plate set-up.
Uneven top to the running part of the mini-gel? Pipette a bit of 0.5-1% SDS solution across the top of the gel solution before it polymerizes. After it polymerizes, just dump out the SDS, before pouring the stack.
I have total street cred! I poured thousands of 0.2mm thick radioactive sequencing gels as a grad student!! w00t!!!!
Ah, but did you screen cDNA libraries using plaque lifts and hybridizations with degenerate oligonucleotides? Or, better yet, screen Î»-phage cDNA expression libraries for sequence-specific transcription factors using plaque lifts and 32P-labeled double-stranded DNA probes, the so-called Southwestern screen? Tons of fun!
but I'm glad there are things like Qiagen RNeasy kits to simplify processing all those tiny bits that I spent hours microdissecting.
So am I. I love Qiagen RNeasy kits with the on-column DNase treatment. The yield isn't always spectacular, but it's the cleanest, most DNA-free RNA I've ever been able to isolate.
I'm not opposed to kits as a matter of principle. It's just that, as with any tool that makes a complicated task more convenient or simple, investigators need to understand how they work.
I've noticed that certain personality types tend to gravitate towards particular technical approaches, within biomedical research. People who do a lot of hard core molecular biology, and people who do a lot of cell/tissue culture (especially with primary, microdissected organs and cell types) tend to be rather obsessive-compulsive. I do both, and I'm about as OCD as one can be, without requiring medication to get out of the house in the morning. I can't comfortably watch Monk, because I'm about 10 synapses away from behaving like that.
In contrast, my experience with electrophysiologist colleagues and friends indicates that they're very spontaneous, seat-of-the-pants types. They're disorganized (by my standards), and may not be able to run a consistent QPCR experiment, but they can build a climate-control chamber for your inverted microscope out of old leaky gel boxes and salvaged electronics bits.
I often tell my new students/trainees that one of the most important steps in casting polyacrylamide gels is cleaning the glass plates. Its my hope that this careful attention to the smallest detail touches every aspect of their lab work. I guess with precast gels I will need a new material for that lesson. Nah, precast gels are too expensive for my needs. I`ll stick to old ways
I often tell my new students/trainees that one of the most important steps in casting polyacrylamide gels is cleaning the glass plates. Its my hope that this careful attention to the smallest detail touches every aspect of their lab work. I guess with precast gels I will need a new material for that lesson. Nah, precast gels are too expensive for my needs. I`ll stick to old ways
You're way too soft on them. Me, I let mine make mistakes and then have them take as long as necessary to trace the problem down (on their own time, of course.) Eventually they figure out that it was a trivial error that could have been prevented by meticulous attention to detail and procedure.
This lesson rarely needs repetition.
I always thought that the pre-cast stuff was the difference between intra-mural NIH labs (unlimited consumables budget hence money no object) and extra-mural NIH-funded Univ labs (Precast? Are you crazy? Do you know what they cost? We have two Masters students' experiments to fund off this grant as well as the postdoc... etc etc.)
When I did a Sabbatical at the NIH a decade back I was amazed (coming from a very scrimping and saving lab culture in the UK) at how much wastage there was. I remember buying a Qiagen miniprep kit which then turned out to be slightly different than the version I was used to. I said I would adjust so that it wouldn't be wasted, but the chief technician just shrugged and told me "don't bother, just get the other one and put that one in a drawer somewhere."
Enjoyed Barn Owl's post. I started off in biochem but found I matched better to his "electrophysiologist phenotype". While the mol cell bio people have the OCD, the electrophys gang have a good share of packrats (like me). Old microscopes are just way too neat to throw out...
PS Making polyacrylamide gels was my first job in a lab - EMBL Heidelberg 1980. Doesn't seem like nearly 30 years.
I'm a graduate student who did a fair amount of work in biomedical laboratory research before cuddling up with numbers instead.
I think the key to it is a balance. Pre-formed gels, Master Mixes and the like make things *faster*, and easier, and less prone to "yeah, today is down the drain because I failed to mix those two, I just layered them on top of each other. Woops".
Similar to high-output computational stuff, new techniques are amazing. But you should do everything by hand first, so at least you know what's going in...so that when what comes out looks wrong, you'll know where to start.
Ah, but did you screen cDNA libraries using plaque lifts and hybridizations with degenerate oligonucleotides?
Damn straight! I loved plating phage more than any task in the lab! I would come in early to pour the plates, so I could do the lifts and get the O/N hybridizations going late the same day.
Well, all right! That's plenty hard core.
I'd be willing to bet that I'm the only surgeon you've ever met who even knows what plaque lifts are, much less plaque lifts and Southwestern hybridizations. ;-)
As a senior grad student who's trained several undergrads, where we use kits, I find it useful to make sure they know what each component is doing, so they still get a useful understanding of what's going on. But I understand the frustration, I've mostly taught physiology students to do hard core molecular and/or cell work.
We just had the first lab here start buying precast gels, I think it's a good idea for the "OK, this is the final replicate, this one's going in the paper for sure" gels rather than the day-to-day jazz.
Also I think the Qiagen kit elution volumes are optimized for concentration and speed rather than yield so I usually increase the incubation time and/or do a second elution with 10-25% of the recommended volume to increase yield.
I've just come off from about eight hours of swearing at SAS and SPSS, so your story hit uncomfortably close to my thoughts on computing while I fiddled with the programming.
What you describe sounds pretty generalizable. We don't spend a full year dissecting a cadaver; we hit the highlights and move on. There's far too much neuro knowledge out there, so we pick what we think is important and move to the next class. Although I'm a decent programmer, most people who use statistical software use the interface and not the command line language.
Aren't we all moving to a world of increasing specialization? My uncle owns a small manufacturing company. A handful of his workers use the foundry, the rest are trained in using increasingly sophisticated manufacturing machines. The old-school casting is becoming a skill set just like the people who make those molds for the kit.
I see your point and think it's a good one, but I can't really tell you how my computer works or how the subway line runs smoothly, and both are vital to my job.
I'm not gonna lie, I kinda miss filling tip boxes. Haven't done that in years.
h, but did you screen cDNA libraries using plaque lifts and hybridizations with degenerate oligonucleotides? Or, better yet, screen Î»-phage cDNA expression libraries for sequence-specific transcription factors using plaque lifts and 32P-labeled double-stranded DNA probes, the so-called Southwestern screen? Tons of fun!
Not so fast old dudes! Some of us youngins can do this too, although I did mine looking for GPCRs. And we found what we were looking for too, but, alas, it turned into the one and only time I got scooped!
OMG ... this is the biology equivalent of neck-bearded UNIX geeks griping that no one knows how to write scripts in PERL anymore.
As an eighth grade science teacher with only an elementary ed degree I will start out by saying I don't understand the details of what your post is about. I do, however, totally get the main idea. Every year my students reply yes when asked if they know how a television works. Then, every year I watch them grow a little more scientific in their thinking when we break it down to what they really know, which is which buttons to press on the remote. They have no idea how any of it works, it just does. Most years a few will be curious enough to hang out and play with a box of wires, light bulbs, batteries and such that I keep, but most of them are only too happy to allow some factory in China to continue handling all those technical details. "Just make the buttons intuitive, please."
One thing needs to be said in favor of old school, and that is the time it takes. It sounds like pouring gels is monotonous work that requires some thought, but for the most part it sounds like there is time to think. Time to think about the procedures for the day, to think about what can go wrong and how it will be resolved, time to put together ideas that have been scattering about in the mind.
On the other hand, pre-cast kits seem to have the workers going straight from their morning commute to recording data, skipping the thinking and wondering part altogether.
On the other other hand, maybe having more time freed up from using the kits allows more time spent looking at and thinking about the actual results of the investigation, which would be a primary goal, I suppose.
Either way, I feel a rant coming on. Someone on earth needs to know how things work. Neither televisions nor gels work by magic, but NCLB and at least one state, Arizona, seem to think so, and unfortunately it seems our young are growing up believing magic potions come in plastic packages and pictures magically appear in boxes.
Electricity is mentioned exactly five times in AZ science standards, grades K - 12. Way back in fourth grade, the kiddies do construct simple circuits*, but jump up to high school and the future keepers of teh internets and the light switches will graduate being able to describe ways in which energy is stored and they will be able to use Coulomb's Law while point distances or charges change. Woot! Millions of Arizona graduates know how balloons stick to walls and ink sticks to paper in the copy machine, but not one can fix the damn thing when it breaks.
Math is just as bad. Along with dozens and dozens of other standards each year, addition and subtraction are mentioned in third and fourth grade, but never again. Fifth graders must recognize that 1 is neither a prime nor a composite, along with 84 other standards that will be on The Test in March. Factoring in school plays and bus drills, the teachers have about one hour for each math standard, to introduce it, teach it, have the kids try it out, and then assess if they learned it. It is scary how many eighth graders cannot add 8 plus 7 but are being tested on their ability to identify the properties of angles created by a transversal intersecting two parallel lines, along with dozens and dozens of other concepts.
Don't even let me get started on the science standards.
What all this boils down to is time. Learning takes time. Thought takes time. Society, culture and technology have changed, yes, but biology hasn't. The human brain needs time to absorb and synthesize new information and to process old information. I believe that time is the underlying factor in the following quotes from above:
I suspect that she doesn't fully understand just how the gel works to separate proteins. She doesn't have that intuitive, visceral understanding gained by fiddling with the components needed to make a polyacrylamide gel and troubleshooting when things go wrong.
For one thing, one's understanding of the science behind a technique can only be strengthened if one has some practical, physical exposure to the technique and how it is done.
Old school techniques are an important learning experience and can do much to help new scientists develop an intuitive understanding of the science behind them, as well as valuable troubleshooting skills.
I believe Orac is grieving about time.
*In Arizona, the kids probably look at pictures of circuits as opposed to actually building them. Forty-ninth out of fifty in spending-- wires, bulbs and batteries don't fit in the budget.
I also write the SAS programming- that way you can tell what's actually been done. I don't know anybody who uses the interface either. Maybe that's just the way I was taught, however.
What I don't do is hand calculate general linear models on pieces of paper, even though I know how to. Why? Because it takes a hell of a long time and the 'puter will do it for me in less than a second with far fewer errors.
Doing everything "old school' is just opening yourself to ridicule. But not knowing how to do things oldschool is ignorant. But then as the glassblowing example shows us this can be reduced to the absurd. Do I have to know how to manufacture paper for instance?
I had the pleasure of taking Techniques in Molecular Biology with a truly excellent professor at Cleveland State. We learned methods old school and new school, and he gave us detailed manuals for each technique that were clear and detailed not just as to how but why you did something.
I know it stuck because while I was reading this post, I could hear his voice in my head, and it made me smile. There are many pleasures in science -- it's nice to be reminded of this.
I have always thought of kits as the lab equivalent of a TV dinner. Preheat the oven, peel back the aluminium foil from the tater tots, and your plasmid prep is done in no time! Useful, to be certain, but no substitute for real home-cooked food when you want to impress someone, or a proper troubleshooting analysis when your gene just refuses to amplify.
I am always interested in learning old-school ways of doing things that I have come to take for granted in kits. Some of this has limits, though. I have a co-worker who refuses to allow me to try water-bath PCR, on the grounds that (a) it takes forever, and (b) once anyone does it, they will refuse to go back to using a thermocycler because the results are so much better. Thus, (c) one takes huge amounts of time doing what usually can be done with the streamlined, modern, and slightly less effective method.
Really, though, prepackaged PCR master mixes?! You can make your own in less than half an hour, and as a bonus have the reagents on hand for when you need to optimise things!
Ahh, there is a gradient gel trick! I may have to give it a try. See, I learned something!
Speaking of things I'd like to give a try, anyone have tips on CsCl DNA preps?
And is there anyway to do a non-kit maxiprep that is endotoxin free?
I'm not at all personally opposed to old skool- I just don't believe there is a glimmer of virtue in suffering for it's own sake.
@Ethan- you totaly cracked me up there.
I teach a lab course for bioengineers, and for the past two years I've been agonizing over pre-cast vs. pouring. Increasingly I see pre-cast gels being used in labs, especially in bioengineering where there are fewer researchers doing gels daily than in other disciplines.
I have to balance the teaching value of the pre-cast gels (which can be significant) with time constraints and acrylamide's nastiness. So far I've stuck with pouring them, but when pre-casts become the norm in our department, that'll probably change. They'll have to make do with the video protocol for pouring their own: http://tdj.berkeley.googlepages.com/SDS-PAGE
I'm a post-doc with ~ 10 years of hard-core molecular biology experience. Just to let you know, Qiagen makes a number of high quality kits, but they are exorbitantly priced. I switched to Promega's PureYield Midiprep kit a while back and it is faster and much cheaper than the equivalent Qiagen kit and the quality of the DNA (OD 260/280) is consistently 1.8-1.9 with high yields and transfection efficiency as good as the EndoFree preps from Qiagen. For gel extraction and PCR purification kits, I recently switched to the QuickClean Kits from GenScript - about 40% the price of the equivalent Qiagen products and quality is just as good. I like Qiagen just fine, but a lot of money in this tight NIH budget climate can be saved by shopping around, getting samples, and talking to colleagues about what they've found works.
Barn Owl said, "In contrast, my experience with electrophysiologist colleagues and friends indicates that they're very spontaneous, seat-of-the-pants types. They're disorganized (by my standards), and may not be able to run a consistent QPCR experiment, but they can build a climate-control chamber for your inverted microscope out of old leaky gel boxes and salvaged electronics bits."
OMG this is so spot-on!
My first advisor was not into actually doing any "advising" -- I was given a box of electronic pieces-parts and told to teach myself how to use the system, and start cranking out data. I had no electronics background, and several parts were broken, but Advisor couldn't tell which -- that was my job, and I eventually got it all fixed and working and cranking out data. I'm still not an electrical engineer, but boy can I trouble-shoot equipment, and have a pretty deft hand with the solder.
To really get how something works, you need a good understanding of the parts. That said, I would agree with the comment about not everyone needing to also be a glassblower (tho' that would be really fun). I mean, we don't require all the micro students to grind their own 'scope lenses!
I still geek out at waveforms and BNC connectors;
Dr Aust is right -- those old working parts might be needed again! And old lab equipment is just too awesomely cool; I'm sure there's a connection in there with the development of steampunk.
However, I beg to differ about the OCD thing -- while tendencies may be rife among mol cel bio folks, they hardly have a monopoly on it. (Pathologists and engineers should proceed with caution should they choose to reproduce together.)
As for gels, when I took Biochem lab the GTA gave us expired kit-gels to use. Only those of us with very careful technique got anything remotely like usable data (I was one), but I kept thinking, "Why don't we just make our own gels up fresh?"
But that would have required too much overseeing on the GTA's part. Then again, xe used to handle polyacrylamide gels bare-handed despite all of us knowing better, and I think too many brain cells had been previously fried by recreational chemicals. What an idiot.
PS Empty pipette tip boxes make fabulous containers for sorting and storing all the electronics bits!
(Pathologists and engineers should proceed with caution should they choose to reproduce together.)
If both are exhibiting symptoms of OCD, I think some such 'cautions' would be a given :)
I'm not gonna lie, I kinda miss filling tip boxes. Haven't done that in years.
Yes! It was always the perfect "I don't feel like working, but I ought to do something semi-productive" task. Plus I could get some good thinking done while hiding out in a corner with headphones and a stack of tip boxes to stuff.
Well, sometimes the old way is better.
But I wouldn't insist you go back to blowing your own glassware, or using your own lungs to draw fluid into a pipette. Some things it's just better to do the modern way.
I'm grateful to electrophysiologist colleagues who save all kinds of bits and pieces-one friend had salvaged the plastic light covers from renovated elevators, and because of the open grid structure, they made excellent platforms for immunostaining cells on coverslips. Moreover, because of the grid structure, the coverslips could be lined up perfectly, and cataloged in diagrams drawn in my lab notebook (OMG SQUEEE!!!). He was more than happy to take them down to the machine shop and cut them to size for me... "See, I KNEW someone would find a use for them! Good thing I saved them." When a pathologist colleague gave me a flow chart for immunostaining and typing mouse sarcomas, I nearly died of OCD happiness.
With this post of Orac's, Coturnix's "lost lab skills" post, and the new Giant's Shoulders classic papers carnival, there's kind of a steampunk science mini-trend going on here....
As a tech who started in the old school, and now works in the new school I saw BAH. I also shake my fist at you kids and yell at you to get off of my lawn. That said I for one am glad that I don't have to spend my time casting gradient gels, or regular SDS-PAGE gels, just pop on in the the device load and go. I can run 4 gels in 2 hours from sample prep to coumassie blue.
In the old days (15+ years ago) I remember when it would take 3 days to get results from your sequencing reaction and we thought that a 400 bp sequence was great. 2 days ago I fedex'd my sample to a company, yesterday I got a 1200 bp sequence back, in the meantime I was able to perform other experiments while I waited. How is this not better??
Orac and others, I get your point about knowing the basic science, but would you rather have me spend hours pouring acrylimide, or do you want the data. I would be surprised if any PI, or lead researcher said, "Oh, its ok, just as long as you understand the basic science surrounding SDS-PAGE, I can wait for the data"
The last molecular genetics class I took was taught by a german gentleman who had done 30,000 F2 crosses by hand for his PhD thesis work. Needless to say, he made us do everything by hand except purifying our enzymes and cycling the PCR mixes. He did threaten to have us sit by two hot water baths for 4 hours, though. >_<
"But I wouldn't insist you go back to blowing your own glassware, or using your own lungs to draw fluid into a pipette. Some things it's just better to do the modern way."
Of course, DLC! The modern, 'graduate student lungs' method is clearly superior.
"WHOA, Old Man, tape?! That is old skool."
I learned to pour gels using tape and I'm now 22. My research professors don't play.
I like Qiagen kits. I think there is something to be said for understanding how these products work, though. Otherwise it's kind of impossible to nail down all the places an experiment could have gone wrong.
Maybe it's just me being uber-Asian, but I see a real value in getting a feel for the "longhand" version of some lab techniques.
By the way, Epicanis, there is a graduate-level scientific glass-blowing course at my institution (and many others, I'm sure). I love our glass shop and have purchased several pretty beaker mugs to support them. Downside, of course, is that blowing glass isn't so great for the environment.
I find that electrophysiologists tend to be instant-gratification types. You get that cell on the rig and you know whether your experiment is working NOW! None of this "do the experiment, load the gel, run it, probe it, and maybe you find out how things went in a day or two" business.
I started doing science around the time electrophysiologists stopped building their amplifiers from scratch. When I first entered graduate school, I'd already switched to electronic calculators (an HP35 that did basic slide-rule functions, had one memory, and cost something like $300), and a postdoc a couple of years older was still using a slide rule. We argued about which was faster, and ended up in a math race. I won, he got a calculator, and I felt a bit like the guy who ran the steam hammer that beat John Henry. I'd never go back to a slide rule, but I still miss them.
We use kits heavily. I certainly don't want students messing around with unpolymerized acrylamide unless it is absolutely necessary, and once you factor in everybody's time, kits are generally cost effective. But you do lose something. When you use a kit, you only have access to the variables that the manufacturer has chosen to provide access to. If the standard conditions don't work because your dealing with some kind of odd situation, you are out of luck. And of course, you lose the ability to do science on a shoestring if your funding falls through and you have to spend time because you don't have the money.
Try doing clinical trials. You get to find out whether it worked in a few years, not a few days.
SDS-PAGE is a core technique for us -- sometimes 50 or more gels a week. We pour the gels 10 or 20 at a time using a little rig that Bio-Rad sells, and have a tiny fortune in plates and combs. It's still a hell of a lot cheaper than precast and the gel formulas are readily optimized for a given protein. If we were not real biochemists and ran say 5 gels a week we'd probably use precasts.