Friday, December 19, 2008

Laboratory Wisdom

Making my usual TAE agarose gel for electrophoresis of some DNA constructs I am attempting to make, I was wondering about some of the 'lab wisdom' that I have received over the years. I think molecular biology is particularly full of superstitions passed from person to person. Probably because it is such usually a 'means to an end' instead of actual science. Specifically I was wondering about the practice on our research floor of cooling the molten agarose to below 70C before addition of Ethidium Bromide (EtBr). The wisdom is that if EtBr is added before the agarose cools you are exposing yourself to vapours containing EtBr. Not good if you know about EtBr.
Quickly, EtBr is a chemical often used to visual DNA in agarose gels. It intercalates between DNA strands and in such an environment is a fluorescent orange/gold colour. Useful, yes. Also it is a mutagen, as determined by the Ames test. According to the Ames test EtBr is somewhere between mothballs and a cigarette in terms of mutagenicity. I guess this is not surprising because of its intimate interaction with DNA.
Lab wisdom is often suspect, as the previous link discusses. Check out some forum discussions on the very topic of EtBr in agarose gels. Scientists apparently like to talk about anecdotal evidence.
My favourite example of bad lab wisdom is about ethanol precipitations of DNA. Most people when performing this common procedure keep the solution of DNA, salt and ethanol on ice or at -20C for a certain time before centrifugation to pellet the precipitated DNA. You can be the biggest annoying know-it-all if, next time you see someone doing this, slap down this reference. This says that this cooling is completely unnecessary and can actually decrease yields. I keep a couple printed out on my bench for just such occasions.
A quick look at the MSDS for EtBr reveals that its vapour pressure is undetermined as is its mutagenic and carcinogenic potential. Sounds like this particular lab wisdom is unfounded but in this case perhaps it is wise.
Any other examples of bad lab wisdom that you happen to know about? Pass it on to the bayblab so that we can correct everyone in a very annoying, know-it-all manner.
[the festive agarose gel was ganked from tim holzer.]


The Doc said...

Curious, this is actually something I've become a little famous for at the Loeb - the 'EtBr is safe' guy. Ethidium bromide is actually used as a drug in people and cattle - it's apparently useful in Sleeping Sickness. Furthermore, it's been used for SS for well over 60 years. No reported cases of cancer as a result (it's called holmium when used as a drug).

It seems it's a good mutagen in one-celled organisms, but multi-cellular organism don't experience the same mutagenic effects.

Even the MSDS's don't list it as being toxic.

"SybrSafe" is acutely MUCH more toxic than EtBr.

Kevin Zelnio said...

We use SybrSafe in our lab. I doubt it is much more toxic the EtBr. I've already reproduced so I don't care so much anyways ;)

I cool down to 55C, but only because anything hotter will warp the trays, not because of the SybrSafe or EtBr. Consequently, I will only autoclave my own water poured straight from the nanopure pump (not from the big jug of np-water). I swear my smeary colony PCR ended when I did that an switched out dNTPs.

rob said...

Alright, here's another one, to see if I can get more myths out of you bayblabbers:
You've probably been told not to stick your finger in an electrophoresis box while a gel is running, because you will get an intense electrical shock. It's not true. If you fill an electrophoresis gel box with buffer, turn it on at say 100V, and stick a single finger in you will not get a shock. Multiple bayblabbers have confirmed this. If you stick in two fingers in the same place in the box such that they are beside each other and equal distance from the electrodes, you will also not get a shock. HOWEVER. if you gradually move those fingers in opposite directions, so that one finger is going towards one electrode and the other finger to the other electrode you will start to get a shock.
The reason is simple: a voltage drop is required between two places on your body in order to have current flow through you, giving you a electrical shock.
Next time a gel is running. Point out the dye front or something to someone and stick your finger into the buffer. (USE A GLOVE DUE TO ETBR). See if they freak.

Anonymous Coward said...

I can confirm the gel thing. Also you might feel slight tingling as you get closer to the electrode. Just make sure not to touch it. The glove is an insulator, so you actually need to take it off to feel anything.

I have a science myth for you: if you leave out 70% ethanol open, does the concentration decrease with time? how fast, and is it linear. Does it stabilize at some percentage?

I wish SOMEBODY would write a post about this...

The Doc said...

Ethidium Bromide:
(inhalation, rat): 0.0118 - 0.1340 mg/L/6H.
(oral,rat): 1503 mg/kg. sts determined to be toxic based on: LC50
(inhalation rat) 0.0472mg/L/1H * 100 (dilution rate)=approximately 4.72mg/L/1H.

Invitrogen's MSDS for SybrSafe:
SYBR Safe dye > 5,000 mg/kg Oral LD50 Rat

This paper:
Suggests that SS is more toxic to bacteria than EtBr.

EtBr, when used as a treatment in cattle and humans, is dosed to about 1mg/kg. You'd need litres of TAE buffer for even a single dose of therapeutic EtBr!

Anonymous Coward said...

On the plus side, TAE/vodka tastes better than eggnog...

Mason Posner said...

I have two. After setting up an SDS-PAGE gel and pouring buffer into the central compartment I use a Hamilton syringe to squirt tank buffer into each well three times. I was told that this clears out the buffer used to make the gel. I have never tested this, as I do not want to waste a poured gel. Is this a lab myth?
Second - I was taught to dial down the quantity on a micropipettor to get an accurate volume. I don't believe this and try not to do it. What do you think?

rob said...

Nice ones.
I actually do both. The first I am sure, at least in a UREA PAGE gel, definitely makes a difference, and also makes it easier to load. (I have accidentally forgotten to do that step many times.)
The dialing down thing, I was told, was just about consistency and some older pipettors have quite a bit of 'slack' in them so maybe that makes sense??

Another one (sort of):
I also remembered the hard way a couple of days ago that laminar flow hoods are not designed to keep vapours away from the user. I guess that's not a myth but I keep thinking it should be like a fumehood because it looks like one. I stank up the tissue culture room real bad. Of course I usually do, but this time I did it with beta merc.

Mason Posner said...

Yah, beta merc fumes in the lab - bad news. Someone in my post-doc lab (before I started working there) dropped and broke a bottle and they had to clear out the entire building for the haz mat crew. I was told that the fumes could be smelled across the street. Since I was not there I have to trust the stories. Still good enough for me to tell every student that if they spill a bottle we would be talking about it years after they graduate.
The ethidium bromide fumes may not be that toxic or carcinogenic, but they always give me a headache and a sore chest when I get a whif. Of course, thinking about the growing tumors in my lungs may be causing some psychosomatic pain.
Love the bat/spider post. Great stuff.

Kamel said...

One bit of gel mythology that I've seen practiced a lot is making fresh ammonium persulfate every time when pouring your own. That stuff will keep in the fridge for quite a while just fine so if you pour plenty of SDS gels you can use the same stock instead of prepping APS every time.

Kevin Zelnio said...

That reminds of a lab myth in our lab. During sequence clean up with magnetic beads we have to make fresh 85% ethanol each time. For some reason my boss insists it matters. I don't really see how it matters to make fresh ethanol dilution or keep a 85% stock around.

I also tested whether it mattered to use autoclaved nano-pure water versus formamide for ABI runs. Nearly identical results with water. That would cut down costs and one less toxic thing in the lab. Formamide is better for longer term storage, but we are just optimizing microsats right now so are not interested in keeping plates after runs.

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