I am a Lab Rat. I work in laboratories, I try to figure out gels and results. I pipette tiny amounts of liquid into various locations. I try to see patterns and shapes and fit things together. This is what I do. I also occasionally venture into the world of Literature and try to find patterns there (but only as a hobby unfortunately).
What I don't do is computers. I can think of over twenty ways to re-phrase the instruction 'look for the comma' (probably over thirty ways if I'm allowed to use the 'synonyms' feature in word) but not one of those ways works for a computer.
Unfortunately there are some things that even Lab Rats need computers for. For instance, searching for a particular domain of DNA, pulling all the results out of a standard BLAST search, and then taking only the relevant information from that. To slightly clarify, BLAST is a bioinformatics programme with a huge database of information about every known and officially sequenced protein and DNA sequence. You type in your sequence (or your name, if you feel bored) and it shows you what proteins it matches on the database. Unfortunately it provides quite a large amount of information about each one, so this is where programming comes in, you tell the computer which bits of the database you want.
Now I did do computer science for IGCSE. I know what pseudocode is, and how to use it, and could probably stab a guess at setting commands up in the right sequence as well. Where I fall apart is the bit after that, translating the pseudocode into computer speak. There are some phrases I quite literally cannot do.
e.g:
IF comma is present
THEN stop
concatenate new information to old (concatenate=attach or add to)
OK. Fine. That works. But how do you say 'comma is present' in computer? The comma is not equal to anything, so that's out. Nor is the comma in relation to anything, it's just a comma in the middle of the string of writing. I have no idea at all how to tell the computer to find me a comma, Perl (which I am using to programme) seems to have no random squiggle that means 'find' or 'look for'.
Another thing that flummoxed me was the concatenation. All the online tutorials showed you exactly how to concatenate, but only if you already knew what the phrases were:
Tutorial: To concatenate x and y type x.=y
Lab Rat: I DON'T KNOW WHAT X AND Y ARE!!! In fact I'm looking for them because I don't know what they are. I want to find that out!;
Tutorial: *is no help at all*
Lab Rat: *Kicks computer, then hold up a large picture of a comma in front of the screen* Just find this, OK? See this picture, find something that looks like this and then give all the writing in front of it to me;
(the semicolon is computer for 'end of line'. I do not know why computers do this when almost every living person uses a full stop).
Computer: *Is not impressed*
I did actually get there in the end, to the surprise and delight of both myself and my supervisor (and probably the computer as well) I managed to get it to do sort of what I wanted. Unfortunately when we looked back over the raw data from our BLAST results we realised that there was a lot more information we wanted, so a lot more code has to be written. And here we hit another problem. The BLAST databases are truly amazing but just not particularly well organised. Some of them have the important information stored under /notes, while others have a separate field called /function. One item we saw even had the full protein function listed under /name. This means that to get all the information we need, we'll have to pull out each of these fields for every single protein, which will provide us with a lot of useless notes that we don't really need.
Lab Rat: Just give me the useful stuff, OK?;
Computer: Variable 'useful' not defined. Random computer squiggles, out of cheese error.
Lab Rat: *gives up on computers*;
Computer: *gives up on Lab Rat*
Some People Juggle Geese
I've spent most of today fighting with computer programmes, but during a brief lull (while pipetting out buffer L2 actually) I suddenly realised that there are an awful lot of odd things that you end up saying in a lab ranging from the slightly weird to the utterly confusing. So here's a little list. All of these have been said (or paraphrased) by me at some point in the last few weeks. And at the time they all made sense...
First, some that are just me being me:
"400 x 5 uh, yeah, that's 2"
"Oh shite, I've lost an order of magnitude"
"I'm going to pretend my lab coat is a trench coat"
"I'm actually beginning to doubt the existence of DNA."
"I can speak to computers!"
A couple of worrying ones:
"Ah. I didn't realise they were that expensive"
"Don't worry! I'm fine! The water just escaped."
"It seems that Agar at 50 degrees is not enough to remove fingerprints."
"Did you just hear the lid explode?"
And last, the vaguely baffling:
"My daisy is on the ice-box."
"Spot on seventy at a random twiddling of the twiddle."
"Shoogle it violently then go as fast as possible."
My favourite phrase is probably the "My daisy is on the ice-box" It has a slightly Monty Python-ish air to it.
First, some that are just me being me:
"400 x 5 uh, yeah, that's 2"
"Oh shite, I've lost an order of magnitude"
"I'm going to pretend my lab coat is a trench coat"
"I'm actually beginning to doubt the existence of DNA."
"I can speak to computers!"
A couple of worrying ones:
"Ah. I didn't realise they were that expensive"
"Don't worry! I'm fine! The water just escaped."
"It seems that Agar at 50 degrees is not enough to remove fingerprints."
"Did you just hear the lid explode?"
And last, the vaguely baffling:
"My daisy is on the ice-box."
"Spot on seventy at a random twiddling of the twiddle."
"Shoogle it violently then go as fast as possible."
My favourite phrase is probably the "My daisy is on the ice-box" It has a slightly Monty Python-ish air to it.
When you don't know who to trust...
To determine how much DNA is present in our DNA extractions, we use a neat little machine called a Nanodrop. This tells us how much DNA is present by giving us the absorption spectrums at A260 and A280 Abs. As DNA absorbs ultraviolet, this tells us what concentration of DNA we have.
It also tells us how contaminated our sample is. The ratio of the 260/280 should ideally be between 1.8 and 2. Any lower and there is significant protein contamination, and higher and there's probably lots of salt or something in there.
Or so we thought. And so quite a few of the references and pages seemed to suggest. Until, of course, we got to the Wikipedia article. This tells us, in no uncertain terms, that it takes a relatively large amount of protein contamination to significantly affect the 260:280 ratio, and even provides a little table to show that. It provides a citation link as well, which I can't get to. I've tried Google and Pubmed until I went round in circles, but no one wants to give me that paper. At least not for free :(
So who do we trust? The wikipedia article has a solid-looking table, whereas in most of the other things we read it was just a throwaway line. On the other hand "most of the other things we've read" includes the instruction manual for the machine, which should know what it's talking about. And I have yet to read the wikipedia citation.
On reflection, I think I will disbelieve Wikipedia this time. At any rate, it hardly matters because we still don't actually have any DNA.
Ah well. At least I'm getting paid for it =D
It also tells us how contaminated our sample is. The ratio of the 260/280 should ideally be between 1.8 and 2. Any lower and there is significant protein contamination, and higher and there's probably lots of salt or something in there.
Or so we thought. And so quite a few of the references and pages seemed to suggest. Until, of course, we got to the Wikipedia article. This tells us, in no uncertain terms, that it takes a relatively large amount of protein contamination to significantly affect the 260:280 ratio, and even provides a little table to show that. It provides a citation link as well, which I can't get to. I've tried Google and Pubmed until I went round in circles, but no one wants to give me that paper. At least not for free :(
So who do we trust? The wikipedia article has a solid-looking table, whereas in most of the other things we read it was just a throwaway line. On the other hand "most of the other things we've read" includes the instruction manual for the machine, which should know what it's talking about. And I have yet to read the wikipedia citation.
On reflection, I think I will disbelieve Wikipedia this time. At any rate, it hardly matters because we still don't actually have any DNA.
Ah well. At least I'm getting paid for it =D
Vaguely Spiritual
I was walking to the lab this morning, when I was stopped by a vaguely hippy-like woman handing out pamphlets. I always feel a bit sorry for people handing out pamphlets as they mostly get ignored, so I took one and decided to take a look at it. It was a religious one 'Does God care about suffering and if so why does it exist' or something like that.
My religious views can best be currently discribed as 'Single and Searching' so I decided to have a look through it. Before I go on to disect the thing though, I will make a quick disclaimer that unless your religious beliefs involve deliberately hurting people then I will respect them. I have no issue of any peoples of any religion, especially not christianity which I rather like because the Christian God actually had a go at being human for a while, which seems to me a sensible and charitable thing to do.
Religion is a touchy subject, but here I go anyway:
The Pamphlet
The first issue adressed was, unsurprisingly, does God exist. Unfortunately rather than going for the reasons I would have chosen (gap between the Mind and the body, the brain in a vat hypothesis, no fixed truth etc) they went for the Arguement from Design. Namely, everything is amazing, the world is so perfect therefore it must be designed. They even used Paley's wristwatch arguement, they went as far as to use the damn eye as well. This made me angry (actually it made me dissolve into giggles, but I should have been angry). Firstly because the whole eye arguement was debunked way back by Darwin, and secondly because if the eye is (as they claim) "Designed so that no camera could have done better" why are so many people wearing glasses? Was there a shortage of perfect eyes? Do only some people get them?
It had a nice phrase about the bible as well "No book has such credentials for historical accuracy". I'm still thinking about that and it baffles me no matter how I look at it.
My favourite bit though was the paragraph about the human 'unique blood system'. I can only presume they mean 'unique' in it's traditional sense of 'possessed by all vertebrates.' The quick science-babble about the blood system was, though, correct. Simplified but correct, which I don't mind at all. They just seemed to draw the oddest conclusions about it.
After all the waffle about the design of the earth, I was expecting it to go downhill from there. Surprisingly, it actually went uphill, dragging itself out of the shady science and giving a reasonable bit about free will. No doubt it would have annoyed the philosophers as much as the bad science annoyed me, but as a lay-person I didn't spot too many blinding errors.
Interestingly the picture of Adam and Eve in Eden had her handing over what was obviously and distinctly a pear. Not an apple. I've heard boths figs and bananas postulated as the actual fruit (my vote is with figs, personally) but never pears before.
After the free will part though, it seemed to take a bit of a fall again. Apparently, we are in the Last Days before the fall. This is because of the Wars, earthquakes and diseases that have been recently appearing in great quantities. It doesn't seem to have occured to the author that Wars, Earthquakes and diseases have always been happening in great quantities pretty much forever in human history. Seriously though, in English history (which I know most about) it's very hard to find a period of more than about 30 years without a war, disease, or natural disaster happening somewhere. Think of the plague, which wiped out almost 1/3 of the population.
It then sort of leaves the rails a bit and heads into lala land. Alright, probably not entirely fair, but I was a bit fed up with it at this point (the wristwatch arguement was still annoying me). The last section was talking happily about after judgement day, for a quick summary read through the last section of 'The Last Battle' by C.S Lewis, because what they were saying was pretty much word for word from the bit where Narnia is destroyed. All the good people will go through into this amazing 'new world', the sick will be better, the dead will come back to life, and presumably the space will be infinate because there are going to be an awful lot of people floating around. Oddly enough it never mentions what happens to be bad people, presumably we get to stay on whats left of the earth which, given the population will have gone down considerably, probably won't be too bad an option. We'll still get sick and die though.
They also mentioned who would be chosen as 'good' although they were remarkably cagey about it. I thought I was in the clear at first because all it was talking about was 'brotherly love' stuff and I'm quite a nice person. However at the end it sneaked in a clause about being and living 'under gods rule.'
Which, as I eat shellfish, means I'm stuffed. :(
My religious views can best be currently discribed as 'Single and Searching' so I decided to have a look through it. Before I go on to disect the thing though, I will make a quick disclaimer that unless your religious beliefs involve deliberately hurting people then I will respect them. I have no issue of any peoples of any religion, especially not christianity which I rather like because the Christian God actually had a go at being human for a while, which seems to me a sensible and charitable thing to do.
Religion is a touchy subject, but here I go anyway:
The Pamphlet
The first issue adressed was, unsurprisingly, does God exist. Unfortunately rather than going for the reasons I would have chosen (gap between the Mind and the body, the brain in a vat hypothesis, no fixed truth etc) they went for the Arguement from Design. Namely, everything is amazing, the world is so perfect therefore it must be designed. They even used Paley's wristwatch arguement, they went as far as to use the damn eye as well. This made me angry (actually it made me dissolve into giggles, but I should have been angry). Firstly because the whole eye arguement was debunked way back by Darwin, and secondly because if the eye is (as they claim) "Designed so that no camera could have done better" why are so many people wearing glasses? Was there a shortage of perfect eyes? Do only some people get them?
It had a nice phrase about the bible as well "No book has such credentials for historical accuracy". I'm still thinking about that and it baffles me no matter how I look at it.
My favourite bit though was the paragraph about the human 'unique blood system'. I can only presume they mean 'unique' in it's traditional sense of 'possessed by all vertebrates.' The quick science-babble about the blood system was, though, correct. Simplified but correct, which I don't mind at all. They just seemed to draw the oddest conclusions about it.
After all the waffle about the design of the earth, I was expecting it to go downhill from there. Surprisingly, it actually went uphill, dragging itself out of the shady science and giving a reasonable bit about free will. No doubt it would have annoyed the philosophers as much as the bad science annoyed me, but as a lay-person I didn't spot too many blinding errors.
Interestingly the picture of Adam and Eve in Eden had her handing over what was obviously and distinctly a pear. Not an apple. I've heard boths figs and bananas postulated as the actual fruit (my vote is with figs, personally) but never pears before.
After the free will part though, it seemed to take a bit of a fall again. Apparently, we are in the Last Days before the fall. This is because of the Wars, earthquakes and diseases that have been recently appearing in great quantities. It doesn't seem to have occured to the author that Wars, Earthquakes and diseases have always been happening in great quantities pretty much forever in human history. Seriously though, in English history (which I know most about) it's very hard to find a period of more than about 30 years without a war, disease, or natural disaster happening somewhere. Think of the plague, which wiped out almost 1/3 of the population.
It then sort of leaves the rails a bit and heads into lala land. Alright, probably not entirely fair, but I was a bit fed up with it at this point (the wristwatch arguement was still annoying me). The last section was talking happily about after judgement day, for a quick summary read through the last section of 'The Last Battle' by C.S Lewis, because what they were saying was pretty much word for word from the bit where Narnia is destroyed. All the good people will go through into this amazing 'new world', the sick will be better, the dead will come back to life, and presumably the space will be infinate because there are going to be an awful lot of people floating around. Oddly enough it never mentions what happens to be bad people, presumably we get to stay on whats left of the earth which, given the population will have gone down considerably, probably won't be too bad an option. We'll still get sick and die though.
They also mentioned who would be chosen as 'good' although they were remarkably cagey about it. I thought I was in the clear at first because all it was talking about was 'brotherly love' stuff and I'm quite a nice person. However at the end it sneaked in a clause about being and living 'under gods rule.'
Which, as I eat shellfish, means I'm stuffed. :(
PayDay
I have just received my first paycheck. This is quite a big moment for me; for the first time ever someone has given me money in order to carry out science, thus making me (in my eyes at least) a Proper Scientist.
I chose to celebrate this great moment in my lifetime by spinning around very fast on my computer chair making squeaky noises. I still can't quite believe that someone has handed me a substantial amount of money for the most enjoyable six weeks of my holiday.
And the best thing is that due to an accumulation of reasons within the lab (and my long summer holiday) I have been allowed to stay a Lab Rat in September as well.
eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee!
As it would probably be a good idea to put something at least vaguely scientific in this post I tried to look up how autoclave tape is made. Autoclave tape looks exactly like masking tape except that it has fine white lines across it. It is also apparently slightly more sticky. You attach it to anything that you are about to put in the autoclave (which heats things up to very high temperatures in order to sterilize them) and as they are autoclaved the white lines go black. This allows you to tell instantly whether or not something has been autoclaved (and is also handy for holding stuff together).
Wikipedia says this:
"Autoclave tape is an adhesive tape used in autoclaving to indicate whether the correct temperature has been reached for the elimination of all living organisms (typically 121 degrees Celsius).[1]
I chose to celebrate this great moment in my lifetime by spinning around very fast on my computer chair making squeaky noises. I still can't quite believe that someone has handed me a substantial amount of money for the most enjoyable six weeks of my holiday.
And the best thing is that due to an accumulation of reasons within the lab (and my long summer holiday) I have been allowed to stay a Lab Rat in September as well.
eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee!
As it would probably be a good idea to put something at least vaguely scientific in this post I tried to look up how autoclave tape is made. Autoclave tape looks exactly like masking tape except that it has fine white lines across it. It is also apparently slightly more sticky. You attach it to anything that you are about to put in the autoclave (which heats things up to very high temperatures in order to sterilize them) and as they are autoclaved the white lines go black. This allows you to tell instantly whether or not something has been autoclaved (and is also handy for holding stuff together).
Wikipedia says this:
"Autoclave tape is an adhesive tape used in autoclaving to indicate whether the correct temperature has been reached for the elimination of all living organisms (typically 121 degrees Celsius).[1]
Small strips of the tape are applied to the items before they are placed into the autoclave. The tape is similar to masking tape but slightly more adhesive, to allow it to adhere under the hot, moist conditions of the autoclave. The tape typically has diagonal markings containing an ink which changes colour (usually beige to black) upon heating. One such ink contains 30.1% lead thiosulfate, 0.6% magnesium carbonate, 20.1% neocryl B8141, 30.1% ethanol, 22.7% ethyl acetate and 49% ink solids. Unfortunately these percentages add up to more than 100%, so this data is completely bogus. "
Yes. Very helpful. The manufacturers aren't particularly keen on giving the secret away either. This maybe for complicated legal reasons or it may just be that it hasn't occurred to them that anybody would be interested in knowing what autoclave tape is made of.
K
There's a slight problem at the lab at the moment. We aren't getting any DNA. The lovely little DNA extraction protocol is not actually extracting huge amounts of DNA for us, if fact it seems to be producing pretty much no DNA for us.
We've checked through the troubleshooting, and in various technique manuals, and found a couple of modifying features to add to our protocol, which will hopefully make it more productive. Most of them are fairly basic (longer incubation's etc.) but the one that caught my eye was the addition of proteinase K at the denaturing step. When you add SDS (which breaks down all the phage proteins to supposedly release the DNA) you also add this mystery stuff called proteinase K.
Not wanting something so scientific to be shooting over my Magical Event Horizon, I went to take a look at what it was. A protease is a substance that breaks down proteins, and it turns out that this is exactly what proteinase K does, it is far more active when surrounded by SDS as well, so hopefully the combination of the two will pretty much shred all the phage proteins releasing all the DNA (as I said, phages are nothing but DNA and protein).
One interesting thing I did find though is that proteinase K was extracted from a fungus (called Tritirachium album Limber). It's fairly deadly as well, it can pull apart all sorts of different proteins. It makes sense for a fungi to possess a powerful proteinase like this as fungi are saprotrophic feeders; they release the enzymes to digest their food outside of their actual bodies and then just absorb all the nutrients left behind.
I thought that the name 'proteinase K' might turn out to have a fairly interesting backstory (why K?) but it is, in fact, spectacularly boring. It was named Protienase K due to its keratin hydrolyzing activity which, seeing as it hydrolyses (breaks down) a large number of proteins seems fairly arbitrary. Biologists have very little imagination sometimes.
If you have a desperate longing to get hold of some proteinase K you can buy it here.
It is fairly expensive though; £39.00 for 2ml.
We've checked through the troubleshooting, and in various technique manuals, and found a couple of modifying features to add to our protocol, which will hopefully make it more productive. Most of them are fairly basic (longer incubation's etc.) but the one that caught my eye was the addition of proteinase K at the denaturing step. When you add SDS (which breaks down all the phage proteins to supposedly release the DNA) you also add this mystery stuff called proteinase K.
Not wanting something so scientific to be shooting over my Magical Event Horizon, I went to take a look at what it was. A protease is a substance that breaks down proteins, and it turns out that this is exactly what proteinase K does, it is far more active when surrounded by SDS as well, so hopefully the combination of the two will pretty much shred all the phage proteins releasing all the DNA (as I said, phages are nothing but DNA and protein).
One interesting thing I did find though is that proteinase K was extracted from a fungus (called Tritirachium album Limber). It's fairly deadly as well, it can pull apart all sorts of different proteins. It makes sense for a fungi to possess a powerful proteinase like this as fungi are saprotrophic feeders; they release the enzymes to digest their food outside of their actual bodies and then just absorb all the nutrients left behind.
I thought that the name 'proteinase K' might turn out to have a fairly interesting backstory (why K?) but it is, in fact, spectacularly boring. It was named Protienase K due to its keratin hydrolyzing activity which, seeing as it hydrolyses (breaks down) a large number of proteins seems fairly arbitrary. Biologists have very little imagination sometimes.
If you have a desperate longing to get hold of some proteinase K you can buy it here.
It is fairly expensive though; £39.00 for 2ml.
I'm in less of a sciency mood...
...because I've just started watching Firefly. wow. such amazing characters. Such amazing character interactions. Such an awesome mix of sci-fi and western without clogging it with unnecessary romance. Seriously though, is there any other sci-fi movie where the pilot is in a loving and stable marriage? Or where explosions in space happen to the accompaniment of awesome guitar music rather than actual scientifically-incorrect explosions?
On the lab front, we are busy troubleshooting. The DNA extraction technique is extracting tiny amounts of DNA and the task today is to find out why. Which means going through the whole process all over again (it takes about six hours) extracting aliquots every time we so much as pick up an eppindorf and try to find out where exactly our DNA is going.
...
They never have problems like this on Serenity.
On the lab front, we are busy troubleshooting. The DNA extraction technique is extracting tiny amounts of DNA and the task today is to find out why. Which means going through the whole process all over again (it takes about six hours) extracting aliquots every time we so much as pick up an eppindorf and try to find out where exactly our DNA is going.
...
They never have problems like this on Serenity.
Agarose gel
Today's task is running gels, to see if our DNA extraction technique managed to extract DNA. Halfway through measuring out the agarose for the gel base I suddenly realised that I had very little idea of how these gels actually worked. Lab Rat job of the day therefore, was to find out more about them (hopefully in order to prevent further embarrassment of the NrdG kind).
Agarose gels
Agarose gel electrophoresis is a basic method used to separate RNA and DNA of different sizes. The gel itself is made up of an agarose matrix (formed from agarose, ethidium bromide and TAE; Tris-acetate EDTA. More on those later) with little wells at the top to add the DNA. As DNA is negatively charged, you can run an electric charge across the gel causing all the DNA to migrate down it.
The agarose and TAE make up a matrix. The TAE acts as a buffer, keeping the agarose at a constant pH. The agarose makes the gel more difficult for the DNA to run through; which allows the DNA to separate. Small fragments can get through relatively easily while larger fragments take a lot longer.
Ethidium bromide is used to visualise the gels. It fluoresces under UV light when intercalated into DNA, allowing you to visualise any band containing ~20ng of DNA under a UV lamp. The problem with ethidium bromide is that it intercalates with any DNA, including the DNA on (say) your skin. This makes it a powerful carcinogen, so it's a good idea to wear gloves and coats while handling it.
As well as length, the distance moved by the DNA is also affected by its conformation. Linear fragments sort nice smart bands (ideally) while circular DNA just forms a sort of elongated blob. Because of this, circular DNA fragments (i.e from plasmids or viruses) are usually cut up using restriction enzymes. As we're just seeing if we've got any DNA at the moment, we haven't bothered cutting it up into pieces, we're just running the circular DNA from our viruses.
All fun stuff :)
Agarose gels
Agarose gel electrophoresis is a basic method used to separate RNA and DNA of different sizes. The gel itself is made up of an agarose matrix (formed from agarose, ethidium bromide and TAE; Tris-acetate EDTA. More on those later) with little wells at the top to add the DNA. As DNA is negatively charged, you can run an electric charge across the gel causing all the DNA to migrate down it.
The agarose and TAE make up a matrix. The TAE acts as a buffer, keeping the agarose at a constant pH. The agarose makes the gel more difficult for the DNA to run through; which allows the DNA to separate. Small fragments can get through relatively easily while larger fragments take a lot longer.
Ethidium bromide is used to visualise the gels. It fluoresces under UV light when intercalated into DNA, allowing you to visualise any band containing ~20ng of DNA under a UV lamp. The problem with ethidium bromide is that it intercalates with any DNA, including the DNA on (say) your skin. This makes it a powerful carcinogen, so it's a good idea to wear gloves and coats while handling it.
As well as length, the distance moved by the DNA is also affected by its conformation. Linear fragments sort nice smart bands (ideally) while circular DNA just forms a sort of elongated blob. Because of this, circular DNA fragments (i.e from plasmids or viruses) are usually cut up using restriction enzymes. As we're just seeing if we've got any DNA at the moment, we haven't bothered cutting it up into pieces, we're just running the circular DNA from our viruses.
All fun stuff :)
Magical Event Horizon
One concept I vaguelly mentioned yesterday, while going through the DNA extraction protocol, was that of the Magical Event Horizon. I love this concept (which I first read in a Terry Pratchett book but anyway) which works on the Arthur C. Clarke quote that "sufficiently advanced technology is indistinguishable from magic".
The idea is, therefore, that everyone understands things up to a point. That point is the Magical Event Horizon (or MEH). For me, for example, bacteriophages and calculus are well within the MEH, relativity is sort of dancing along the line and the inner workings of computers are far, far over my Magical Event Horizon.
It's a neat little concept. Anything that you personally sort of feel to yourself works by 'magic' (like digital cameras. I have no idea how those work) goes over the MEH, everything you can explain stays within it.
The idea is, therefore, that everyone understands things up to a point. That point is the Magical Event Horizon (or MEH). For me, for example, bacteriophages and calculus are well within the MEH, relativity is sort of dancing along the line and the inner workings of computers are far, far over my Magical Event Horizon.
It's a neat little concept. Anything that you personally sort of feel to yourself works by 'magic' (like digital cameras. I have no idea how those work) goes over the MEH, everything you can explain stays within it.
DNA Extraction
Todays task is extracting DNA from our T4 phages that we've spent the last two weeks growing and nurturing and caring for. To make this easier, we're using a ready-made Kit called QIAGEN (which I keep trying to pronounce qui-gon :D ). QIAGEN basically provides all the material and gives an idiot guide as to how to use it, which means that Lab Rat's task of the day involves going through said protocol and working out what is actually happening in each step.
First though, another quick notice from the Department of the Very Obvious:
---
DNA Extraction
1.Grow the phages on agarose gel. Agar gel is a gelatenous substance from seaweed that contains both agarose and agaropectin. Agaropectin contains lots of acidic sidegroups (containings sulfur and various other things like pyruvate). Agar is usually used for gels as it's cheaper, but for DNA extraction you can't risk any of the acidic agaropectin loitering around as it stops the extraction enzymes from working. Instead you use just pure agarose.
2.Isolate phages. This is done by peeling off the top layer of agarose and using a centrifuge to spin it down to the bottom, leaving the phages in the supernatant (the liquid left behind after centrifuging). Push the supernatant through a couple of filters to remove anything else (bacteria mostly) and you're left with a pure phage solution.
3.Now the DNA extraction can start. The first instruction in the kit is 'add buffer L1'. Buffer L1 is a clever mixture of various different enzymes and buffer solutions which breaks down any bacterial RNA or DNA that might be left in your solution.
4.Next buffer L2 is added. This precipitates out the phage particles; essentially it clumps all the bacteriophage's together making them easier to extract.
5.Centrifuge to collect the phage. The centrifuge is the big fast-spinning machine that pellets all the phages down into a neat little, well, pellet. Very useful machine, and it would be even more useful if ours worked properly :(
6.Resuspend the phage in buffer L3. I suspect this is just a growth medium, to turn the phage pellet into a phage suspension.
7.Add buffer L4. Buffer L4 contains the well known SDS (which comes up very often in various extractions). SDS is a detergent which essentially breaks all the proteins down. As viruses pretty much just consist of proteins and DNA this means that the only whole thing left in the test-tube at this point is the DNA. Still quite a way to go though, so here's a diagram of a centrifuge:
8. Add buffer L5, mix and centrifuge. It does not actually say, but i suspect L5 is precipitating the proteins that have just been cut up. The centrifuge will then pellet the proteins down to separate them from our DNA.
9. Equilibrate a QIAGEN-tip 20 by adding buffer QBT. This is where we hit the Magic Event Horizon (MEH) at top speed. I have no idea what is in the buffer or exactly how a QIAGEN-tip works, which is probably a good thing for copyright in general. You can get to QIAGEN to find out more about it by going here: QIAGEN
10. Allow the residue from step 8 to flow through the QIAGEN tip. One thing I do know about the tips is that they contain resin. The resin traps the DNA on it allowing the rest of the phage to wash through (essentially this will just be any liquid medium, as all the proteins have technically been removed by the centrifuge in step 8)
11. Wash the QIAGEN tip with buffer QC. This washes the DNA (which is trapped on the filter) removing any last impurities.
12. Collect DNA with buffer QC into a clean tube. The QC in some way (Magical Event Horizon fast approaching) allows the DNA to flow through the filter and into the new tube. Finally the DNA! We now (should) have a solution containing nothing but phage DNA. Just got to collect it.
13. Precipitate the DNA by adding isopropanol. Isopropanol is another old favourite, it just clumps DNA, making very very hard-to-see pellets. You've probably guessed by now but the next stage is: centrifuging, to collect the pellet.
14. Wash pellet with ethanol. Ethanol removes any residual salt as there will be some magnesium and various others in the DNA (DNA has an overall negative charge due to the phosphate backbone which collects positively charged salts). Washing involves adding ethanol, shaking very gently and then (surprise surprise) centrifuging to pellet the DNA again and remove all the ethanol.
15. Allow DNA to dry and redissolve in buffer. Buffer just keeps the DNA happy and stops it disintegrating.
And that's it! We now have a little glass bottle containing a solution of pure DNA.
Science really is just like cookery =D
First though, another quick notice from the Department of the Very Obvious:
- Do not try to do DNA extractions when hung-over
---
DNA Extraction
1.Grow the phages on agarose gel. Agar gel is a gelatenous substance from seaweed that contains both agarose and agaropectin. Agaropectin contains lots of acidic sidegroups (containings sulfur and various other things like pyruvate). Agar is usually used for gels as it's cheaper, but for DNA extraction you can't risk any of the acidic agaropectin loitering around as it stops the extraction enzymes from working. Instead you use just pure agarose.
2.Isolate phages. This is done by peeling off the top layer of agarose and using a centrifuge to spin it down to the bottom, leaving the phages in the supernatant (the liquid left behind after centrifuging). Push the supernatant through a couple of filters to remove anything else (bacteria mostly) and you're left with a pure phage solution.
3.Now the DNA extraction can start. The first instruction in the kit is 'add buffer L1'. Buffer L1 is a clever mixture of various different enzymes and buffer solutions which breaks down any bacterial RNA or DNA that might be left in your solution.
4.Next buffer L2 is added. This precipitates out the phage particles; essentially it clumps all the bacteriophage's together making them easier to extract.
5.Centrifuge to collect the phage. The centrifuge is the big fast-spinning machine that pellets all the phages down into a neat little, well, pellet. Very useful machine, and it would be even more useful if ours worked properly :(
6.Resuspend the phage in buffer L3. I suspect this is just a growth medium, to turn the phage pellet into a phage suspension.
7.Add buffer L4. Buffer L4 contains the well known SDS (which comes up very often in various extractions). SDS is a detergent which essentially breaks all the proteins down. As viruses pretty much just consist of proteins and DNA this means that the only whole thing left in the test-tube at this point is the DNA. Still quite a way to go though, so here's a diagram of a centrifuge:
8. Add buffer L5, mix and centrifuge. It does not actually say, but i suspect L5 is precipitating the proteins that have just been cut up. The centrifuge will then pellet the proteins down to separate them from our DNA.9. Equilibrate a QIAGEN-tip 20 by adding buffer QBT. This is where we hit the Magic Event Horizon (MEH) at top speed. I have no idea what is in the buffer or exactly how a QIAGEN-tip works, which is probably a good thing for copyright in general. You can get to QIAGEN to find out more about it by going here: QIAGEN
10. Allow the residue from step 8 to flow through the QIAGEN tip. One thing I do know about the tips is that they contain resin. The resin traps the DNA on it allowing the rest of the phage to wash through (essentially this will just be any liquid medium, as all the proteins have technically been removed by the centrifuge in step 8)
11. Wash the QIAGEN tip with buffer QC. This washes the DNA (which is trapped on the filter) removing any last impurities.
12. Collect DNA with buffer QC into a clean tube. The QC in some way (Magical Event Horizon fast approaching) allows the DNA to flow through the filter and into the new tube. Finally the DNA! We now (should) have a solution containing nothing but phage DNA. Just got to collect it.
13. Precipitate the DNA by adding isopropanol. Isopropanol is another old favourite, it just clumps DNA, making very very hard-to-see pellets. You've probably guessed by now but the next stage is: centrifuging, to collect the pellet.
14. Wash pellet with ethanol. Ethanol removes any residual salt as there will be some magnesium and various others in the DNA (DNA has an overall negative charge due to the phosphate backbone which collects positively charged salts). Washing involves adding ethanol, shaking very gently and then (surprise surprise) centrifuging to pellet the DNA again and remove all the ethanol.
15. Allow DNA to dry and redissolve in buffer. Buffer just keeps the DNA happy and stops it disintegrating.
And that's it! We now have a little glass bottle containing a solution of pure DNA.
Science really is just like cookery =D
Things I've learnt today...
If you're writing a report, don't write the discussion in five minutes straight. And don't print immediately after writing the discussion. And don't hand the report in without checking you've written everything you want to write.
uuurgh...
I had lots of interesting things to put in that discussion as well. It was going to be a good discussion.
Other things I've learnt:
:(
Good news though: We have phage! They grew, with no contamination, which means that tommorrow we get to slice them open and take their DNA out. YAY!!
uuurgh...
I had lots of interesting things to put in that discussion as well. It was going to be a good discussion.
Other things I've learnt:
- Don't waste time looking at random sites when you should be writing reports
- Don't do this so often that you end up writing the report the morning it's due
- Don't miss breakfast to write a report
- The pope is a catholic
- Bears really do shit in the woods.
:(
Good news though: We have phage! They grew, with no contamination, which means that tommorrow we get to slice them open and take their DNA out. YAY!!
Contamination!
I came in this morning, all bright and ready for phage propagation (harvesting phages from plates is actually quite fun) and what did we find? All the plates from yesterday were bad. To make a phage plaque you plate out a smooth 'lawn' of bacteria onto an agar plate and then pour phage over it. Where one phage lands it kills and multiplies until it forms a little clear spot or 'plaque' on the plate. (you can't see the phage plaques very well in the picture, but you can see the bacterial lawn)This morning, all our plates had little fuzzy marks on them. Which is fine, could be phages except...so did the control plate. The control plate had no phage on it at all, it was meant to have just the bacteria but itstead it too had little fuzzy marks.
*sigh*
Without good controls you can't trust your results. At first we thought it was just badly grown host until my supervisor noticed that the tryptone we'd been working with (tryptone broth is one medium that you can grow the bacteria in) was cloudy. Not good. If a substance that is usually clear starts looking cloudy then it means it's got bacteria in it.
Our plates were all contaminated. The fuzzy marks were phage, bad host and/or another bacterial contaminant. Basically we have to do all of yesterdays work all over again today and we need a lot more media (agar for plates, tryptone for bacteria, etc).
Guess who gets to make up all the new media?
I do love working in labs. But it can sometimes be the more frustrating thing on earth. And media pouring is just mindnumbingly boring and faintly awkward. Which is why, of course, the lab rat gets to do it.
How to look like an idiot...
A fairly embarrassing thing happened yesterday afternoon. My supervisor's PI (the one who owns the lab and is in charge of all the research) came by to see how we were doing and he asked me about my bioinformatics project. My bioinformatics project involves looking at bits DNA from our bacteriophage and trying to work out what it does, using all sorts of clever internet search programs (mostly BLAST actually).
The conversation went something like this:
PI: So, hows the work going?
Lab Rat: Fine. I'm pretty sure about the first sequence, it's got a fairly obvious NrdG domain, and it's really conserved with other NrdG domains. I reckon it's a NrdG.
PI: Great. So what does an NrdG domain do? Why have we got one in the phage?
Lab Rat: *frantically scans notes*. Uh, an NrdG domain, uh, ribonucleotide reductase, uh, class III anaerobic ribonucleotide reductase. Um. assembles deoxyribonucleotides.
PI: ...
Lab Rat: I'll look it up.
So my first task this morning was heading over to PubMed. For those not in the know, PubMed is the worlds most wonderful search engine, which scans all the science reviews and papers and can usually find the one you want. For those who are interested, here is a brief overview of what NrdG domains are:
The conversation went something like this:
PI: So, hows the work going?
Lab Rat: Fine. I'm pretty sure about the first sequence, it's got a fairly obvious NrdG domain, and it's really conserved with other NrdG domains. I reckon it's a NrdG.
PI: Great. So what does an NrdG domain do? Why have we got one in the phage?
Lab Rat: *frantically scans notes*. Uh, an NrdG domain, uh, ribonucleotide reductase, uh, class III anaerobic ribonucleotide reductase. Um. assembles deoxyribonucleotides.
PI: ...
Lab Rat: I'll look it up.
So my first task this morning was heading over to PubMed. For those not in the know, PubMed is the worlds most wonderful search engine, which scans all the science reviews and papers and can usually find the one you want. For those who are interested, here is a brief overview of what NrdG domains are:
NrdG
NrdG is a domain in a protein, which basically means that it is an area of the protein with a recognisable structure. Search engines such as InterProScan are designed to look for these domains in proteins and show you where they are. You can then (if you do your research properly) go to PubMed and find out what the domain does.
One of the most important tasks in a cell is the replication of cellular DNA. DNA is built up from small subunits, or monomers, known as deoxyribonucleotides. In order to replicate the DNA the cell must have enough of these monomers to make a new strand of DNA. However most cells do not replicate all the time, at different times in their life cycles they will need different concentrations of deoxyribonucleotides inside the cell.
Enter ribonucleotide reductase: This controls the concentration of DNA monomers within the cell. It works by using oxygen to generate a tyrosyl radical (basically just a very reactive molecule, radicals are very reactive and have been shown to play an important role in catalysis). The tyrosyl radical then catalysis the reaction, producing new DNA monomers. Control of ribonucleotide reductase therefore, controls the amount of DNA monomers.
The problem with this is that some bacteria don't use oxygen. These are known as anaerobic bacteria. Without oxygen, they cannot generate the tyrosyl radical, which is vital for the reaction to occur. So how do they manage it? This (finally) is where my NrdG domain comes in. The NrdG domain contains a class III reductase, which is an anaerobic reductase. Instead of using oxygen to create a tyrosyl radical, it uses a different system to produce a glycyl radical. Still a radical (so it still has high reactive power) but made from a different molecule (glycerol rather than tyrosine).
The golden question is of course, what is this doing in my phage? Bacteriophages don't make DNA, they invade bacteria and use that bacteria to make DNA for them. Also, my bacteria is a lytic phage which means that it's only in the bacteria for a short amount of time before killing it. (unlike lysogenic phages, which hang around in the bacteria for long lengths of time. Lysogenic phages often carry bits of DNA which their bacterial hosts will find useful).
For a phage to carry a ribonucleotide reductase is not, however, such a bad idea. The host for my phage is a bacteria which can survive in both aerobic (with oxygen) and anaerobic (without oxygen) conditions. It is plausible, therefore, that the phage carries with it a gene that helps the bacteria to make DNA, especially in anaerobic conditions, when most cellular processes tend to be slower anyway. That way the phage can get its DNA made up and packaged as quickly as possible and then break out of the bacteria.
Feel free to ask any questions in the comments :)
One of the most important tasks in a cell is the replication of cellular DNA. DNA is built up from small subunits, or monomers, known as deoxyribonucleotides. In order to replicate the DNA the cell must have enough of these monomers to make a new strand of DNA. However most cells do not replicate all the time, at different times in their life cycles they will need different concentrations of deoxyribonucleotides inside the cell.
Enter ribonucleotide reductase: This controls the concentration of DNA monomers within the cell. It works by using oxygen to generate a tyrosyl radical (basically just a very reactive molecule, radicals are very reactive and have been shown to play an important role in catalysis). The tyrosyl radical then catalysis the reaction, producing new DNA monomers. Control of ribonucleotide reductase therefore, controls the amount of DNA monomers.
The problem with this is that some bacteria don't use oxygen. These are known as anaerobic bacteria. Without oxygen, they cannot generate the tyrosyl radical, which is vital for the reaction to occur. So how do they manage it? This (finally) is where my NrdG domain comes in. The NrdG domain contains a class III reductase, which is an anaerobic reductase. Instead of using oxygen to create a tyrosyl radical, it uses a different system to produce a glycyl radical. Still a radical (so it still has high reactive power) but made from a different molecule (glycerol rather than tyrosine).
The golden question is of course, what is this doing in my phage? Bacteriophages don't make DNA, they invade bacteria and use that bacteria to make DNA for them. Also, my bacteria is a lytic phage which means that it's only in the bacteria for a short amount of time before killing it. (unlike lysogenic phages, which hang around in the bacteria for long lengths of time. Lysogenic phages often carry bits of DNA which their bacterial hosts will find useful).
For a phage to carry a ribonucleotide reductase is not, however, such a bad idea. The host for my phage is a bacteria which can survive in both aerobic (with oxygen) and anaerobic (without oxygen) conditions. It is plausible, therefore, that the phage carries with it a gene that helps the bacteria to make DNA, especially in anaerobic conditions, when most cellular processes tend to be slower anyway. That way the phage can get its DNA made up and packaged as quickly as possible and then break out of the bacteria.
Feel free to ask any questions in the comments :)
Phages
So...as this is my first post, I might as well start with what I'm working on. Bacteriophages.
Bacteriophages (usually shortened to phages) are viruses that attack bacteria. They tend to look like little lunar landing modules; essentially they 'land' on the bacteria, inject in their DNA and replicate it using the bacterial DNA replication proteins. They propagate inside the bacterial cell and then, when finished, burst out of it. This kills the bacteria and leads eventually to nice little plaques forming on an agar plate.
The most interesting thing about phages is that they attack bacteria exclusively. This is because bacteria are prokaryotic, and have a very different internal structure too eukaryote cells (like yours, or mine, or that of a sheep etc). Prokaryotes have no nucleus, and no proper chromosomes, usually just circular loops of DNA. They also use different types of replication proteins and have very different cell surface proteins (although some bacteria can mimic host cell proteins, mostly for infection purposes).
This means that the best thing about phages is that they can potentially be used to treat bacterial infections. Once the phages reach the site of infection they can multiply, kill off the bacteria, and then quietly depart (I am still not exactly sure what actually happens to phages once they've killed all the bacteria, but what I do know is that they are not dangerous in any way). This is a wonderful idea, saves the use of antibiotics, but also carries a slight social stigma with it, after all technically it involves giving people viruses. Even though these viruses don't actually do people any harm at all.
Then I read this:
phagetherapy
For those unwilling to trawl through the link, the basic information is that not only is phage theory a good idea in practise, it is also an idea that works. While Western Europe has been fiddling around with antibiotics and coping with MRSA and other resistant bugs, there are labs in Eastern Europe that are already researching and testing phage therapy. They have very small labs and the conditions and funding are both fairly appaling but the work is being done. This, I think, was my favourite sentance:
"Each soldier in the Georgian army carried a spray-on phage cocktail which they used to disinfect their wounds "
The stuff is actually out there, it works and it's relatively safe (more on that in a bit), why isn't it being researched and tried more often?
Safety:
There are very few side affects associated with phage therapy. As far as I know there's really only one although it is potentially fairly hazerdous. Phages work by killing the bacteria (as mentioned before) which unfortunately leaves a large amount of bacterial debris. In some cases, this is picked up on by the immune system which assumes it's under massive bacterial invasion and over-reacts. This produces what's known as a 'cytokine storm' where lots of immune chemicals (e.g cytokines) are released causing a result similar to anaphalaxis (which is what happens with asthma and allergic reactions).
This is not, however, a novel side effect. It already happens with some antibiotics. And phage therapy has so many more benefits. Bacterial resistance happens less often, and as the phages mainly use the virulence factor surface proteins (the proteins which produce the things that make you feel ill) of bacteria as markers for landing and invasion, resistant strains will be less dangerous. Phages can also evolve with the bacteria, changing to adapt to resistant mutants.
That's why I decided to work on phage therapy.
Bacteriophages (usually shortened to phages) are viruses that attack bacteria. They tend to look like little lunar landing modules; essentially they 'land' on the bacteria, inject in their DNA and replicate it using the bacterial DNA replication proteins. They propagate inside the bacterial cell and then, when finished, burst out of it. This kills the bacteria and leads eventually to nice little plaques forming on an agar plate.
The most interesting thing about phages is that they attack bacteria exclusively. This is because bacteria are prokaryotic, and have a very different internal structure too eukaryote cells (like yours, or mine, or that of a sheep etc). Prokaryotes have no nucleus, and no proper chromosomes, usually just circular loops of DNA. They also use different types of replication proteins and have very different cell surface proteins (although some bacteria can mimic host cell proteins, mostly for infection purposes).
This means that the best thing about phages is that they can potentially be used to treat bacterial infections. Once the phages reach the site of infection they can multiply, kill off the bacteria, and then quietly depart (I am still not exactly sure what actually happens to phages once they've killed all the bacteria, but what I do know is that they are not dangerous in any way). This is a wonderful idea, saves the use of antibiotics, but also carries a slight social stigma with it, after all technically it involves giving people viruses. Even though these viruses don't actually do people any harm at all.
Then I read this:
phagetherapy
For those unwilling to trawl through the link, the basic information is that not only is phage theory a good idea in practise, it is also an idea that works. While Western Europe has been fiddling around with antibiotics and coping with MRSA and other resistant bugs, there are labs in Eastern Europe that are already researching and testing phage therapy. They have very small labs and the conditions and funding are both fairly appaling but the work is being done. This, I think, was my favourite sentance:
"Each soldier in the Georgian army carried a spray-on phage cocktail which they used to disinfect their wounds "
The stuff is actually out there, it works and it's relatively safe (more on that in a bit), why isn't it being researched and tried more often?
Safety:
There are very few side affects associated with phage therapy. As far as I know there's really only one although it is potentially fairly hazerdous. Phages work by killing the bacteria (as mentioned before) which unfortunately leaves a large amount of bacterial debris. In some cases, this is picked up on by the immune system which assumes it's under massive bacterial invasion and over-reacts. This produces what's known as a 'cytokine storm' where lots of immune chemicals (e.g cytokines) are released causing a result similar to anaphalaxis (which is what happens with asthma and allergic reactions).
This is not, however, a novel side effect. It already happens with some antibiotics. And phage therapy has so many more benefits. Bacterial resistance happens less often, and as the phages mainly use the virulence factor surface proteins (the proteins which produce the things that make you feel ill) of bacteria as markers for landing and invasion, resistant strains will be less dangerous. Phages can also evolve with the bacteria, changing to adapt to resistant mutants.
That's why I decided to work on phage therapy.