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Recombinant DNA For The Home Hobbyist
Dr. Zowie writes: " Scientific American 's "Amateur Scientist" column
this month tells how to amplify and isolate DNA chains in your kitchen, using the
tried-and-true Polymerase Chain Reaction technique.
Use it
for massively parallel computing experiments; to ID friends, pets, and favorite houseplants; or to help eliminate epidemics. But what'll happen when
enterprising teenagers start playing with plasmids and recombinant
DNA?" I love articles that remind you that one of the ingredients it recommends playing with is a nasty mutagen. Interesting that PCR has become so common that all it takes is a hundred dollars and a dark room!
O.K, so it's a fun idea and all, but i thought i'd clarify that the article is simply instructions on Amplifiying and Sequencing DNA, not changing or cloning people :) The two are just a little too diferent :)
Syllable : It's an Operating System
The article neglects to mention that PCR is covered by several patents. For instance, here's the fine print from an advertisement for Clontech's AdvanTaq DNA polymerase:
"Purchase of Advantage PCR reagents is accompanied by a limited license to use them in the Polymerase Chain Reaction (PCR) process for research in conjunction with with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front license fee, either by payment to Perkin-Elmer or as purchased, i.e., an authorized thermal cycler."
Roche holds most of the relevant patents on PCR, though they recently lost one of them as a result of a long, ugly lawsuit against Promega. For details, see this page at about.com. Perkin-Elmer holds a bunch of patents on machines for performing the thermal cycling step.
Fiddling with PCR in your own home is arguably an "experimental use" (i.e. you just want to see if it works) and therefore permitted under patent law, but don't make any commercial plans to Make DNA Fast.
I would like to see some open source designs for some hardware to automate putting base pairs together. Then hook it up to a computer and have another team write a gcc- cross compiler that compiles genes.
Step three is to write a really high level language so we could write a script such as:
PromDate
{
Blond
Blue Eyes
5 foot 10 inches
36-24-36
}
Feed it to the compiler and out come a set of chromosomes.
Then no geek would ever be dateless to the prom again.
As it is, the closest thing to this PCR I ever did was bake yeast bread. That took three tries because the first two times I killed the yeast when I made the water too hot.
For thouse who want to try this at home, drop in the local toy store and look in the "science" area. You may find a $10 kit to do just this.
I've found most scientific supply places are a pain to deal with when you want something delivered to a home address. Sigma (out of St Louis) called me 3 times to find out why I was ordering $3 worth of tubing.
In such a case, it would seem to me that 'overrated' would be a better classification.
Either way, if someone truly is karma whoring, then what exactly is being accomplished by telling them?
You can't tell someone's intentions by the content of their post. Maybe they really are unfunny and mistaken? Those are observations that can be made without the 'karma whore' label (which seems to be attached to just about anyone but the people who use the label itself).
Just my $.02
- Jeff A. Campbell
- VelociNews (http://www.velocinews.com)
- Jeff
Well, lego has a few things that might make the accuracy easier. I was just looking through a catalog because I think I'm going to start to get into robotics via lego. :)
Okay, I started out doing a much longer list, and sort of flailed. And I know I'm offtopic, but heck, legos are just so darn cool. Anyway, I was thinking about something similar to this, and I decided you'd need four things to get high positional accuracy out of legos. Two of them are above, and the other two are lego blocks with a bearing inset for smooth, tight rotation, and metal drive shafts. If you have some good resistance behind it, those plastic shafts are definitely going to twist and flex, which you absolutely don't want.
"You're right," Fisheye says. "I should have set it on 'whip' or 'chop.'"
. .on a bloody knife I found in a dumpster in Brentwood CA. It identifies some guy named Orinthol James something or other.
___
My research partner (read wife) and I have been recombining DNA in for years. We even did it in the kitchen once, however, the bedroom is more suited to our techniques. So far the results look promising, but I'll keep you posted if there are any changes.
I'd rather you do it wrong, than for me to have to do it at all.
James?? James!! Quit amplifying your sister's DNA and eat your dinner! NOW, young man!
For those of you who might actually be compelled to try this at hope, here's a tip to save a little money. Scientific supply companies will frequently provide free samples for disposable equipment, like pipette tips or eppendorf tubes -- and there are a lot of scientific supply companies out there, so lather, rinse, repeat.
I had a (poorly funded) professor who kept her lab going for weeks with freebies. Sometimes she even managed to weasel out some more expensive items, like a free sample of Taq polymerase.
In fact this entire issue of Scientific American is really worth reading. Discussion on life in the Universe, A huge report on The Human GENOME and the race to patent your body. Incredable stuff.
In Soviet Russia...michael would be rotting in Siberia!
of a study done my several of my friends and I at our lab up in Boulder, Co.
Basically, it was an experiment in the neural synaptic responses produced by the oral ingestion of Delta-9 TetraHyrdaCanabinol via several metalic media heated on an electronic thermal amplifier.
results: when you smoke weed via the "hot knives" method...you get really fucking danked!!
FluX
After 16 years, MTV has finally completed its deevolution into the shiny things network
"It is seldom that liberty of any kind is lost all at once." -David Hume
0100100000100000011010010111000010000001100001001
And how do I go about getting this DNA? Can I use this as an excuse in case my significant other catches me one night in the den?
Significant Other: Honey, what the hell are you doing? Lotion? A videotape of 'Girls Gone Wild Part IV'??
Me: Uhhh.. I'm just getting some DNA, baby. It's all in the interest of science, trust me.
I mean, yeah, you could just extract some plasma but that wouldn't be fun.
Small potatoes make the steak look bigger.
When I was an undergraduate in the late '80s I remember the big fuss when our university received its first PCR machine when PCR was viewed as a high-tech futuristic technique, much as microarrays are seen today. And now look at PCR -- amateurs can do it at home! I wonder if ten years from now microarrays will be cheap enough for amateurs to play with.
Sorry--hit submit by mistake.
p cr_notes.htm
This article is seriously a waste of time. Why would anyone want to sit around for 3 hours moving tubes from water bath to water bath like grad students did 15 years ago?
All the supplies needed would cost hundreds and hundreds of dollars. A vial of amplitaq gold polymerase (the enzyme I use) alone costs $130. The dNTPS cost probably another $80, the agarose costs $100, the EtBr costs $30, the primers cost $40, etc. If someone packaged just the reagents you would need for a few PCR reactions together, it wouldn't be so bad.
The reason why this would be a waste of time is because the article makes it sound a lot easier than it really is, and I can imagine how much work it would take to get this going in the kitchen, and in the end you will have only replicated something idiotically routine by lab standards. But if someone really wants to try this, a few suggestions: first, the first time you put the tubes in the 94C water bath, make sure you leave it there for about 5 minutes, not just 1 as on the remaining steps. Second, the temperatures of the water baths are critical. Don't let the first water bath get over 95C or your polymerase will degrade too rapidly. Make sure the second water bath is at EXACTLY the right temperature for the primers you're using, within 2 degrees. The third water bath is not as critical. Another thing, when you're making your DNA template from a cheek scraping, a few mls of water will probably be too much. Try several concentrations of template DNA. Also, the drop of mineral oil has nothing to do with damaging the polymerase when freezing; it's to prevent refluxing inside the tube. One other thing is that you should not try to add the MgCl2 to the buffer yourself if you're using a coffee stirrer instead of a pipette, but you can buy buffer with MgCl2 already in it.
Optimizing the reactions can also be a bitch, but here's a URL to help with that:
http://www.immunologists.net/sxprotocols/molbiol/
This reminds me of Boris Vian's song, La java des bombes atomiques , which described some amateur tinkerer making atomic bombs in his garage...
(Quick English translation below for the french-impaired)...
La java des bombes atomiques
My uncle, a famous tinkerer
used to make, as an amateur
some atomic bombs
Without ever learning anything
he was a real genius
when it came to practical works
He locked himself all day long
in his workshop
to make his experiments
And in the evening,
he came back home,
and explained it all to us.
To make an A-bombs,
children, believe me,
it's really a piece of cake.
The detonator question
is solved is a quarter hour
it's the one we put aside.
And for the H-bomb,
it's not much harder,
but one thing bothers me,
is that the bombs I make
only have a action radius
of only three meters fifty.
There's something wrong there,
I'm going back right now.
He worked at it for days
trying, with love,
to improve the yield.
When he ate with us,
he wolfed down his soup
We saw to his appearance
that he fell upon a hard part
but we dared not say anyhing.
Then one evening, during the meal,
here he sighs, and starts shouting:
As I'm getting older,
I see better
that my brain is failing
it ain't a brain anymore
it's like béchamel sauce
It's been months and years
I've tried to increase my bomb's
yield, and I never noticed
that the only thing that matters
it's the place where it falls down.
There's something wrong there,
I'm going back right now.
Knowing that success will be close,
all the great heads of state
came to visit him.
He received them and excused him
that his shop was so small.
But as soon as they were all in,
he locked-them up,
telling them be nice!
And when the bomb went off,
of those people nothing remained.
My uncle, in front of the result,
didn't chicken-out
He played the dummy
In front of the court
Before the jury,
he mumbled
Gentlemen, it's a horrible bad luck
But I swear in front of God
That in my soul and conscience
That by destroying those crooks,
I am convinced of having
Served my countryu.
They were embarrased,
So they sentenced him,
then they pardoned him.
And in reward, the country
elected him head of the government.
--
Here's my mirror
I'm not sure it's such a good idea for Sci Am to suggest that individuals play with carcinogenic/mutagenic reagents at home. I mean, I am growing increasingly leery of all the shit I use in the lab (I'm a molecular biologist) and its cumulative effects on my body. I'm only 26 and I don't want to have flipper babies because I breathed in some fumes once or twice.
That said, this is an informative article, because it serves to "de-mystify" the science of DNA manipulation. That's important because IMHO, a lot of the public's fear of this technology stems from not understanding it. Of course, that portion of the public probably doesn't read Sci Am...
Freedom: "I won't!"
... why not try reading something by the inventor of the reaction itself, Kary Mullis? He wrote a book called Dancing Naked in the Mind Field, which is mostly just a large collection of stories, opinions, and anything else he wanted to put in there. As you should be able to see immediately, this guy is no ordinary scientist. As for the book, I loved it, and most of you probably would too. There's a lot of stuff in there that will make you challenge how you've been thinking about science, such as the chapter about HIV and AIDS. The whole thing is very not-politically correct, like the chapter on his experiences with LSD and related substances, and that makes it more fun to read. Pick up a copy, you won't regret it.
u iry.asp?isbn=0679774009
the book at bn.com - http://shop.barnesandnoble.com/booksearch/isbnInq
cos its fun and it teaches you something. It may be routine for lab folks but it isnt for the average /. reader (who's not a biochemist). plus, more people working with DNA can lead to some serious breakthroughs, given enough time. /. readers working in hitech anyway...and tinkering is a natural reaction with most of us.
A coupla $100K labs avaiable to the general public would be nice, but isnt going to happen. A few hours or days and a $1000 budget is ok for most
they have packed most of the stuff together..check out the link at the bottom of the article.
Here's some software to help design your primers. http://www.alkami.com/primers/refdsgn.htm Primers run about $50 a pop and will last about 10-20 rxn's, depending on if you're using a coffee straw or a PipetMan. Find em here: http://www.stratagene.com/pcr/pcrprimers.htm or http://www.lifetech.com You can also buy many reagents (phenol, chloroform, B-mercaptoethanol isopropanol, glycogen, NaAc, etc) relatively inexpensively, and do a decent DNA extraction.
But isn't the purpose of the Doomsday machine lost if you keep it a secret!
Here are also two ways to destroy Ethidium Bromide chemically. One is uses reagents that are harder to get (but does a better job), while the second uses ordinary bleach (but the destruction is less complete).