Bought a couple books
Jason Morrison
"Shoestring Biotechnology"
http://biotechinstitute.org/programs/publications.html#shoestring
"Co-published with the National Association of Biology Teachers (NABT), this laboratory manual provides low-cost, practical, teacher-tested activities for high-schools and two-year colleges to support the integration of biotechnology into the classroom. Shoestring Biotechnology: Budget-Oriented High Quality Biotechnology Laboratories for Two-Year College and High School provides the basis for the teacher professional development that the Institute conducts around the nation. "
and
"Biotechnology: A Comprehensive Curriculum Guide for a One Semester Course at the High School (grades 11-12) or Community College Level"
http://www.biotechinstitute.org/resources/MossBiotechGuide.html
"The question from many teachers who wish to start a biotechnology course is "where do I begin?" This manual offers high school and community college educators with a detailed "road map" on how to start a biotechnology course at the high school or community college level. It was written by former biotechnolgoy executive turned high school teacher who started and taught a biotechnology course for five years. The manual provides details about curriculum, resources (texts, labs, Internet) used, and equipment required. It is designed to provide teachers a foundation upon which they can build and customize their own course curriculum."
I'll let you know how they are! If anyone else in the Boston area is interested in checking them out, I'll bring them to our next meetup.
-Jason
--
Jason Morrison
jason.p....@gmail.com
http://jayunit.net
(585) 216-5657
Lim
worked at Lincoln High right?) give a talk at the S.B. 4.0. Pretty
compelling stuff.
On Nov 1, 12:06 pm, "Jason Morrison" <jason.p.morri...@gmail.com>
wrote:
>
> "Shoestring Biotechnology"http://biotechinstitute.org/programs/publications.html#shoestring
>
> "Co-published with the National Association of Biology Teachers (NABT), this
> laboratory manual provides low-cost, practical, teacher-tested activities
> for high-schools and two-year colleges to support the integration of
> * provides the basis for the teacher professional development that the
>
> and
>
> "Biotechnology: A Comprehensive Curriculum Guide for a One Semester Course
> at the High School (grades 11-12) or Community College Level"http://www.biotechinstitute.org/resources/MossBiotechGuide.html
>
> "The question from many teachers who wish to start a biotechnology course is
> "where do I begin?" This manual offers high school and community college
> educators with a detailed "road map" on how to start a biotechnology course
> at the high school or community college level. It was written by former
> biotechnolgoy executive turned high school teacher who started and taught a
> biotechnology course for five years. The manual provides details about
> curriculum, resources (texts, labs, Internet) used, and equipment required.
> It is designed to provide teachers a foundation upon which they can build
> and customize their own course curriculum."
>
> I'll let you know how they are! If anyone else in the Boston area is
> interested in checking them out, I'll bring them to our next meetup.
>
> -Jason
>
> --
> Jason Morrison
Mackenzie Cowell
I think it would be interesting to compare the regulations he has had to deal with (apparently very few) to get his high school lab booted up and running with what would be required to start an undergraduate lab at a college with the same equipment and curriculum. Furthermore, his lab and his iGEM team have gotten a fair amount of press, and none of it has alarmed any regulators.
Mac
Julie E Norville
DIYbiologists have
in coupling with high schools in order to create supplemental science
education
programs to teach more experimental biological science to high school
students.
Possibly DIYbiologists could team with a local high school biology class and a
biology teacher to create a summer biolab on the high school campus. The
DIYbiologists involved could be sure to "design-for-safety" in choosing what
experiments they will do. The DIYbiologists can also provide a fair bit of
transparency by posting their experiments and results online. The students
involved will gain a hands on feel for biology. Since DIYbio aims to greatly
reduce the costs and barriers to entry for biological experimentation,
the high
school involved need not necessarily be a rich high school in a rich
neighborhood.
Garage biology or biology done in secret can bring up visions of
Frankenstein or
Dr. Moreau. On the other hand open pedagogical bioscience as an objective has
the potential to both opens young minds to science and would establish
DIYbiology as a service organization, without limiting the capacity of its
members to do good science and good engineering. This also potentially makes
DIYbiology a good partner for NSF and other granting organizations that make
outreach part of their missions.
Julie
Bryan Bishop
> Possibly DIYbiologists could team with a local high school biology class and a
> biology teacher to create a summer biolab on the high school campus. The
> DIYbiologists involved could be sure to "design-for-safety" in choosing what
> experiments they will do. The DIYbiologists can also provide a fair bit of
> transparency by posting their experiments and results online. The students
> involved will gain a hands on feel for biology.
High school teams have competed in iGEM before, so yes, it's been done
and is being done. If you need some materials for starting a high
school team, I might be able to dig up some stuff. The technical
information isn't sparse, though I would recommend you talk with some
iGEM regulars for promotional materials or something to show how
exciting and interesting it all is before somebody shuts down the
biology classrooms.
> Garage biology or biology done in secret can bring up visions of
> Frankenstein or Dr. Moreau.
Geeze, kinda like breeding done in secret. Terrible! Disasterous! You
mean people breed in private!? Bring back the swinging sixties, or
something. Oh wait, they don't want that either. I am very confused.
:-)
Mackenzie Cowell
I agree completely. We need a practical project to begin with - perhaps optimizing (if necessary) and transforming an interesting gene in biobrick format into ADP1.
I would like to get ADP1 and try this experiment at NUBlabs. Once we get the infrastructure and process fine-tuned, I think it would be appropriate to build it into a high school curriculum. Or should we get a class involved right now?
I'm going to begin by tracking down a source of ADP1 and contacting the professor that Jason pointed us at who has ADP1 genes in biobrick format. If anyone is interested in helping me - please email me!
Mac
Julie E Norville
CSAIL, has a contact at a high school (potentially a high school in Newton)
that he and Erik Demaine have worked with in the past. I think he is out of
the country now, but he might be able to help us get in contact with
the people
there, if desired. Also, I think SynBERC (through the Student Leadership
Committee) may be interested in sponsoring some interactions between
DIYbio and
high school students or teachers, in the $1000 to $2000 range. This
may require
writing a proposal that describes what the money is being used for.
Harris Wang had some interesting ideas for synth/diybio projects that might be
appropriate for DIYbiologists in a high school environment whereas the
FlashLabs BioWeatherMap (http://blog.diybio.org/) could easily be appropriate
for any DIYBiologist anywhere, and would probably attract little controversy.
On Wednesday Nev. 12th and Wed Nov. 19 12-1pm, I would like to organize a
"Build-Your-Own" Pipette workshop during the Synthetic Biology Working Group
Lunch at MIT. During this time we can try to develop a liquid measuring
system, as has been suggested in the past by Pam Silver and Tom Knight, and do
so in a way that is much less costly ($20-$50) than the standard lab setup
(~$1300, not including tips for liquid measurement). Perhaps we can publish
the results/blueprints online or in an open source journal. This is the
standard measurement instrument for biology, but is quite expensive to operate
outside a standard research lab.
Mackenzie Cowell
What other ideas are out there for homebrew pipettes? It seems like exploiting surface tension / capillary action might enable extremely simple mechanisms.
What about different tared masses of filter paper, each calibrated to absorb a useful volume? Would they be too unreliable? And I guess no one wants bits of filter paper floating around in their reaction mixes. String? Why don't we just freeze everything and work with solid shavings? :)
Other ideas?
Mac
Meredith L. Patterson
increments. They're not reusable, though, and you'll need a sharps
container.
A friend of mine and I are putting together plans for a lab robot on
servos -- think of an X/Y/Z array kind of like one of those claw
games, but replace the claw with a manipulator on servos that can move
in 3-space. We'll use a Wiimote as a controller (see
http://todbot.com/blog/2007/10/25/boarduino-wii-nunchuck-servo/ for a
simple example). We'd like to be able to use it as the basis for a
multichannel pipette array, though we haven't worked out how to
measure out liquids yet.
--mlp
Mackenzie Cowell
This page about alternative molecular biology equipment that the Journal of Chemical Education published an article back in 1991 about building your own pipette - can anyone chase it down and mail it to me?
Journal of Chemical Education, Vol. 68, No. 4, Apr 1991
Thanks!
Mac
Jason Morrison
On Wednesday Nev. 12th and Wed Nov. 19 12-1pm, I would like to organize a
"Build-Your-Own" Pipette workshop during the Synthetic Biology Working Group
Lunch at MIT.
Mackenzie Cowell
John Cumbers
Does anyone have experience making and purifying their own enzymes? I presume it involves tagging the protein and then running it through a column to purify it? What other ways are there to purify enzymes? Are any of these protocols DIYable? For example if we can come up with a way to make our own restriction enzymes then we could also then start experimenting with ways to keep them active at room temp/over time etc. e.g. encapsulate into a wax bead etc.
Just some thoughts,
John
John Cumbers, Graduate Student
NASA Ames Research Center
Mail Stop 239-20, Bldg N239 Rm 373 Moffett Field, CA 94035, USA.
cell +1 (401) 523 8190, fax +1 (650) 604-1088
Graduate Program in Molecular Biology, Cell Biology, and Biochemistry
Brown University, Box G-W Providence, RI, 02912, USA
Jim H
More than willing to contribute. Since I have purified every RE and
most of the ME's in the IVGN catalog (I wouldn't doubt they're still
selling some Acc- I or Sca-I produced in a massive batches in '95).
No one was tagging proteins back then and I would be surprised if they
do now. It's just a pain in the a** and the chealating resins
ridiculously expensive. Only one we did with a HIS tag was Proteinase
K.
The easy way, over just about any ion exchange bed, is to find you
enzyme looking for banding patterns in fractions across the column
developed with simple salt gradients. You also look for non-specific
endonuclease by looking at smearing of unrecognized plasmid sequences
and pool away from these.
I can help anyone who's interested in doing this.
>
> >>> On Wednesday Nev. 12th and Wed Nov. 19 12-1pm, I would like to organize a
> >>> "Build-Your-Own" Pipette workshop during the Synthetic Biology Working
> >>> Group
> >>> Lunch at MIT.
>
> >> --
> >> Jason Morrison
>
> >>http://jayunit.net
> >> (585) 216-5657
Guido D. Núñez-Mujica
only extracted from fungus, so I assumed it has post transcriptional
mods that made it impossible to make it in bacteria.
Jim H
them. I know people who did keep copies, though :-)
Pretty sure Protienase K was cloned into e coli. It was HIS tagged,
so it wasn't a "native" enzyme in any case. But I don't recall which
bug we purified it from.
On Nov 20, 4:16 pm, "Guido D. Núñez-Mujica"
Guido D. Núñez-Mujica
however, with methods like the TruTips, it is less necessary.
Something I just found:
Proteinase K from Tritirachium album Limber. Characterization of the
chromosomal gene and expression of the cDNA in Escherichia coli.
Gunkel FA, Gassen HG.
Institut für Biochemie, Technische Hochschule Darmstadt, Federal
Republic of Germany.
The cDNA and the chromosomal gene encoding proteinase K from
Tritirachium album Limber have been cloned in Escherichia coli and the
entire nucleotide sequences of the coding region, as well as 5'- and
3'-flanking regions have been determined. The deduced primary
translation product consisting of 384 amino acid residues (molecular
mass = 40,231 Da) contains an N-terminal region of 105 amino acids not
present in the mature protein. By analogy to the evolutionary-related
bacterial subtilisins and other serine proteinases it is inferred that
the primary secreted product is a zymogen containing a 15-amino-acid
signal sequence and a 90-amino-acid propeptide. The propeptide is
presumably removed in the later steps of the secretion process or upon
secretion into the medium. The nucleotide-sequence analysis of the
gene and its flanking regions has revealed that the proteinase-K gene
is composed of two exons and one 63-bp-long intron located in the
proregion. Furthermore, a putative promoter sequence and a capping
site have been identified, suggesting that the transcription-start
site is located 103-bp upstream of the ATG initiation codon. To
express the proproteinase-K gene in E. coli, proproteinase-K cDNA was
cloned in a plasmid vector under control of the tac promoter. The
hybrid plasmid pSPPRO, constructed for this purpose, contained the
cDNA coding for proproteinase K [from Ala (-91) to the C-terminal Ala
(279)] fused to the N-terminal-signal-peptide sequence of the
alkaline-phosphatase gene preceded by the tac promoter. E. coli
BMH71-18, harbouring this plasmid, exhibited slight proteolytic
activity when tested on skimmed-milk plates, suggesting that some
fusion proteins were correctly secreted into the periplasm and
processed to the mature proteinase K.
************
Tweaking around with the induction conditions (accounting for rare
codons and folding problems, Rosetta or Origami strains or they DIY
equivalents) and with the sequence, tagging the gene with HIS, maybe
synthesizing the DNA instead of cloning it from mRNA could make make
practical the production of Proteinase K in the garage, maybe in the
adequate conditions is not that painful in the rear.
John Cumbers
Sounds fun, but I will leave it to others to pursue for the time being..
John
John Cumbers, Graduate Student
NASA Ames Research Center
Mail Stop 239-20, Bldg N239 Rm 373 Moffett Field, CA 94035, USA.
cell +1 (401) 523 8190, fax +1 (650) 604-1088
Graduate Program in Molecular Biology, Cell Biology, and Biochemistry
Brown University, Box G-W Providence, RI, 02912, USA
Dan
> So before the biotech companies produced the enzymes that we now buy, people
> used to make them themselves. I know a few labs that make their own taq
> enzyme (now that it is off patent),
>
> Does anyone have experience making and purifying their own enzymes? I
> presume it involves tagging the protein and then running it through a column
> to purify it? What other ways are there to purify enzymes? Are any of
> these protocols DIYable? For example if we can come up with a way to make
> our own restriction enzymes then we could also then start experimenting with
> ways to keep them active at room temp/over time etc. e.g. encapsulate into
> a wax bead etc.
http://nar.oxfordjournals.org/cgi/content/abstract/5/7/2373
A general method for the purification of restriction enzymes
Patricia J. Greene, Herbert L. Heyneker, Francisco Bolivar, Raymond L. Rodriguez, Mary C. Betlach, Alejandra A. Covarrubias, Keith Backman, David J. Russel, Robert Tait and Herbert W. Boyer
Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA 94143, USA
Received April 24, 1978. An abbreviated procedure has been developed for the purification of restriction endonucleases. This procedure uses chromatography on phosphocellulose and hydroxylapatite and results in enzymes of sufficient purity to permit their use in the
sequencing, molecular cloning, and physical mapping of DNA.
might be of interest.
Dan
--
|| Dan || dan[at]dreadportal.com || http://dreadportal.com/ ||
"Reality is that which, when you stop believing in it, doesn't go away."
(Philip K. Dick - How to Build a Universe)
Mackenzie Cowell
enzymes is a fascinating idea. Maybe we could start with taq and use
that to amplify dna for use in testing the gel box 2.0 project.
Jim, what organism do you think would make a safe source for the
proteins (someone in past on this list has talked about isolating taq
from environmental microbes), and what basic equipment and reagents
would be needed to isolate taq?
Cheers,
Mac
Jim H
Funny you should ask. I am moving in with an old colleague and we're
planning on making Taq. Probably just rec e coli, but it's my new
partner who's driving this one. I'll let you know after the first of
the year when we get running.
BTW, taq is not, technically, a restriction enzyme. It's a
Polymerase, or DNA modifying enzyme. You can isolate other DNA
polymerases from "native" bugs, but the advantage of Taq is that it is
thermostable. The names of the RE's tend to come from the "native"
bateria they were originally isolated from i.e. EcoRI= e.coli, Sal-I =
salmonella, etc.
We wouldn't need taq right off the bat for running a simple agarose
gel. Any plasmid + RE would do and that's not too expensive.
Main thing would be to have a centrifuge (or filter, which is more
expensive but better) and a couple chromatography columns. If we have
some substrate DNA (i.e. a plasmid that has a RE cut site and one that
does not), we could fractionate the column and find the desired
activity.
Daniel Wexler
Alec Nielsen
Bryan Bishop
> This would be a huge boon to DIYbio. Enzymes are expensive, and the ability
> to make your own polymerase/REs/ligase would be empowering.
A while back I was doing some writeups on the list for a
"self-replicating" (of sorts) diybio 'reactor'. This was going to
include enzymes and purification processes, plus all of the tools
necessary to do basic experiments. Another factor was a built in
*biological* DNA synthesis mechanism. A few others and I did some
writeups on the experimental procedures that would be needed to
progress to that status, and after a while it started to look like a
PhD's worth of work (at least). Anyway, the ability to say, "here you
go, here's your biology lab in a box" - including organisms, enzymes,
and tools, is a really neat concept. The only thing that wouldn't be
self-produced would be the enclosing container I guess, metals and
glass etc. Protein purification remains a big hurdle IMHO.
Jim H
Most of these RE's were patented by NEB (couldn't find Xba-I, but
here's Nco-I
http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&p=1&u=%2Fnetahtml%2FPTO%2Fsearch-bool.html&r=11&f=G&l=50&co1=AND&d=PTXT&s1=Xba&s2=%22New+England+Biolabs%22.ASNM.&OS=Xba+AND+AN/%22New+England+Biolabs%22&RS=Xba+AND+AN/%22New+England+Biolabs%22)
Most of them are from the late 80's, early 90's so going to expire
soon. I would say all of them are considered "proprietary" or
protected by patents.
I *may* know a few people that *may* have some of these recombinant
plasmids. I'll start digging. Having personally isolated almost
every one of these REs, I can tell you that the procedure is pretty
straight forward and anyone can do it with a blender, a few buffers
and two columns.
On Dec 31, 2:26 am, "Alec Nielsen" <alecniel...@gmail.com> wrote:
> This would be a huge boon to DIYbio. Enzymes are expensive, and the ability
> to make your own polymerase/REs/ligase would be empowering.
>
> For the various BioBrick formats (BBa, Berkeley, Silver, Freiburg, Knight),
> it would be nice to have certain restriction enzymes at your disposal.
> Specifically: EcoRI, XbaI, SpeI, PstI, NotI, BglII, BamHI, XhoI and NheI.
> Does anyone know if any of these enzymes are proprietary, or are their
> sequences all readily available?
>
> A quick Registry search doesn't turn up any of these enzyme-coding
> sequences. We should acquire, standardize and submit them! Where can we get
> the CDSs for this stuff? Does anyone have access to the source organisms
> (besides E. coli), or other engineered strains that have these coding
> sequences? Also, which method of protein purification is most amenable to
> DIYbio?
>
> Inexpensive enzyme availability would help disseminate real gengineering
> capability to the community. Even once DNA synthesis becomes dirt cheap,
> it's conceivable that many DIYers would still do old-fashioned
> 'cut-and-paste' genetic engineering, so this could definitely be worthwhile.
>
> Alec
>
> > Back in 1978 I worked as a tech in Jim Darnell's lab at the Rockefeller
> > Institute. Restriction enzymes for cloning were either not available or
> > extremely expensive to purchase. For our experiments we partially purified
> > our own EcoRI in a single step using a long gel filtration column.
>
> > Jim H wrote:
>
> > Mac,
>
> > Funny you should ask. I am moving in with an old colleague and we're
> > planning on making Taq. Probably just rec e coli, but it's my new
> > partner who's driving this one. I'll let you know after the first of
> > the year when we get running.
>
> > BTW, taq is not, technically, a restriction enzyme. It's a
> > Polymerase, or DNA modifying enzyme. You can isolate other DNA
> > polymerases from "native" bugs, but the advantage of Taq is that it is
> > thermostable. The names of the RE's tend to come from the "native"
> > bateria they were originally isolated from i.e. EcoRI= e.coli, Sal-I =
> > salmonella, etc.
> > We wouldn't need taq right off the bat for running a simple agarose
> > gel. Any plasmid + RE would do and that's not too expensive.
>
> > Main thing would be to have a centrifuge (or filter, which is more
> > expensive but better) and a couple chromatography columns. If we have
> > some substrate DNA (i.e. a plasmid that has a RE cut site and one that
> > does not), we could fractionate the column and find the desired
> > activity.
>
Austin Che
NEB is pretty friendly. They'd probably give you the expression
plasmid if you asked nicely. It's not like any commercial
competitor couldn't easily create such a plasmid if they wanted to
(i.e. the plasmid isn't exactly a trade secret). I believe most of
their expression plasmids are standard (e.g. T7) with the enzyme
sequences all available in REBASE (run by NEB):
http://rebase.neb.com/rebase/rebase.html
http://rebase.neb.com/rebase/rebase.seqs.html
As long as no one here is going to try to purify and sell NEB
patented enzymes, I don't see NEB caring too much. It actually is
probably in NEB's benefit to give away the plasmids. They want
more people to do molecular biology as that's their target
market. Once people realize the difficulties in getting
high-quality enzymes or just get bored of purifying their own
enzymes, they'd just order them from NEB. With the proper choice
of enzymes, I'm not sure it makes economic sense to purify your
own enzyme. It's not that expensive to purchase, e.g. EcoRI
probably costs 10 cents per reaction. For the typical low scale
amateur biologist (e.g. making yeast glow), they might need to run
50 such reactions ($5). I think the purification and QC would cost
more. Maybe the problem is that you can only order enzymes for
hundreds of reactions at a time?
Noah Sachs
As to homebrew production of enzymes, like Taq, wow, there is so much
going on in this thread I don't know where to start.
It depends on your priorities, whether you are looking to save money
or learn something. I don't want to make any assumptions, but making
your won Taq won't save you any money unless you are using a lot of
it. By the time you buy all the stuff for producing an enzyme, it
would be much cheaper just to buy or beg for some enzyme. As a
learning experience, the production of an enzyme is a great lesson,
but secretly.... performing for yourself the quality control assays
that are done on the commercially available enzymes (except for the
hot (radioactive) labeled assays) is a huge learning experience and
should not be missed, it provides a real depth of understanding as to
what makes for a good enzyme of that particular type. Like Star
activity for a restriction enzyme, some may have read about, but it's
valuable to actually see it and then you'll really know it.
But, that being said I can help you make Taq, I know some of the top
guys at making Taq, I can maybe get them to do a lesson on it, maybe
get you a nice cloned version into e.coli that would be super easy to
make Taq from.
Noah Sachs
like in vitro protein expression http://www.neb.com/nebecomm/products/productE6800.asp
that way you wouldn't have to worry as much about purification? I have
been thinking about that as well, and it would take more than a PhD
worth of research to make it, but if you can find something like 30
people or groups to each work on 1 piece of it, then it could be done
in a couple of years.
On Dec 31, 2:34 am, "Bryan Bishop" <kanz...@gmail.com> wrote:
> 1 512 203 0507
Bryan Bishop
> For the ideal "biology lab in a box", were you considering something
> like in vitro protein expression http://www.neb.com/nebecomm/products/productE6800.asp
> that way you wouldn't have to worry as much about purification? I have
> been thinking about that as well, and it would take more than a PhD
> worth of research to make it, but if you can find something like 30
> people or groups to each work on 1 piece of it, then it could be done
> in a couple of years.
Yes, in vitro protein expression is an important component to include,
but the problem with that is the specialty chemicals that have to be
involved.
" PURExpress is a novel coupled cell-free transcription/translation
system reconstituted from purified components necessary for E.coli
translation. Recombinant histidine-tagged aminoacyl-tRNA synthetases
(20), initiation factors (3), elongation factors (3), release factors
(3), ribosome recycling factor, methionyl-tRNA transformylase, T7 RNA
polymerase, creatine kinase, myokinase, nucleoside-di-phosphate
kinase, and pyrophophatase provide the activities required for coupled
transcription and translation, as well as energy regeneration.
Purified 70S ribosomes, amino acids, rNTP's, and tRNA's complete the
system."
So then you have to think about purifying all of *those* dependencies. Yikes.
- Bryan
Jim H
http://www.ambion.com/techlib/basics/translation/index.html
Easy enough to make, as long as you have a willing bunny for a heart
punch. Ok, the bunny doesn't necessarily have to be willing....
> 1 512 203 0507
Daniel Wexler
Jim H
I can speak with certainty that lyophilization is not the answer.
Glycerol stocks work just as well and are more stable for longer
periods of time stored @ -20C. Lyophilized enzymes still need to be
stored @ +4C (but better to keep @ -20C), require optimization of the
solute and are a pain in the arse to rehydrate and whilst maintaining
functionality. I am not saying it won't work with technology today,
but in the late 80's & early 90's we attempted to lyophilize the whole
line of BRL enzymes and it just wasn't a product people wanted, and
for good reason (as stated above). And please don't get me started on
"dried down" enzymes using trehalose. The hype doesn't live up to
performance.
Glycerol stocks are the way to go, now. If there was an
"encapsulated" form, like I have seen in passing somewhere (?), that
might be the ticket.
Daniel Wexler
Daniel Wexler
Jim H
Don't mean to be trite, but I was an associate scientist purifying
enzymes and a supervisor AT Life Tech (BRL) in the Cell Bio and
Enzymes groups for almost 10 years. We did about 30 enzyme preps per
month, so literally thousands of enzymes preps I personally
performed, qualified, analyzed or reviewed. Never saw "star activity"
once. We measured glycerol concentration very carefully, using a
refractometer to within 1/10 of a percentage. It's a very simple
variable to control.
Meredith L. Patterson
lyophilized enzymes and dried E. coli is competitive with glycerol
stocks, so -- in the interest of what would go into a hypothetical
"comprehensive diybio kit" -- what's easiest / cheapest to produce,
and what's easiest / cheapest to ship and store?
--mlp
Jim H
with a good vacuum pump, which would be key. Obviously, preferable to
have an ambient shipping condition. And I concede that lyophilization
is perhaps the best ambient solution we have now. I just know there's
a better way, but don't have the answer.
On Jan 1, 4:09 am, "Meredith L. Patterson" <clonea...@gmail.com>
wrote:
> lyophilized enzymes and dried E. coli is competitive with glycerol
> stocks, so -- in the interest of what would go into a hypothetical
> "comprehensive diybio kit" -- what's easiest / cheapest to produce,
> and what's easiest / cheapest to ship an
Daniel Wexler
"Alw21I, BpiI, Bsp68I, BspTI, Eco32I, Eco91I, EcoRI, Hin6I, HinfI, Mph1103I, Mva1269I and NcoI are especially sensitive to the high glycerol concentration in the reaction mixture".
Daniel Wexler
Bryan Bishop
> As to homebrew production of enzymes, like Taq, wow, there is so much
> going on in this thread I don't know where to start.
Don't forget purification. :-)
http://www.protocol-online.org/prot/Molecular_Biology/Protein/Extraction___Purification/Purification_of_Specific_Proteins/
http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=3098
The original site no longer exists, so I'm copying the protocol here
for the sake of prosperity and interest.
=============
Home-made Taq Polymerase Purification
I also have the bacteria containing the clone. It appears to produce
lots of Taq and is quite stable.
The proceedure takes 4 days start to (15 000 units of Taq) finish.
The Taq also appears very stable and reliable. I made 15 mls a year
ago and it still works fine.
Reference: Engelke, D. R. et al. Anal. Biochem. 191:396-400 (1990).
Pluthero, F. G. et al. NAR. 21:4850-4851 (1993).
Day 1
1) Inoculate two 2L flasks of TB/amp (500ml) with 15ml of an overnight
of Taq bugs. These volumes may be scaled down.
2) Grow to an OD600 of 0.6 (approx mid log)
3) Add IPTG to 0.5mM (0.119gm/litre), grow o/n but not for more than 16hrs.
Day 2
N.B: All the following proceedures should be carried out on ice or at least 4°C.
4) Collect cells by centrifugation (3.5K / 15 mins / 4°C) and
resuspend in 40ml buffer A.
5) Add an equal volume of buffer B (45-50ml) and incubate at 75°C for
1hr, with periodic mixing.
6) Centrifuge ( 8K / 15mins / 4°C).
7) Add 1.86mg of KCl / ml of supernatant.
8) Aliquot equal volumes of supernatant into each of 2 x 250ml
centrifuge tubes (preferably conical) containing 75ml of washed Sigma
DP-1 cation exchange resin (packed volume). This should be washed 2 x
with sterile water and 4 x with ice cold Buffer B.
9) Vortex tubes well and incubate on a shaking platform (30mins / 4°C).
10) Centrifuge (approx 3K / 2min / 4°C) to pellet resin and discard
supernatant.
11) Wash resin 4 x with 100-200ml of ice cold buffer B, remove
supernatant by aspiration.
12) Elute 3 x with one packed bed volume of ice cold buffer C.
13) Add 30gm (NH4)2SO4 / 100ml of eluate while stirring rapidly.
N.B: At this point it is of great advantage if you use conical or
round bottomed tubes. i.e. 50ml tubes for the 8x50 rotor. Prior to
this the sample may be handled in 250ml centrifuge bottles for ease of
use.
14) Centrifuge at 12-15Krpm for 10mins at 4°C.
15) Resuspend pellet (weakly translucent) in 25-35ml of buffer C.
16) Dialise 2 x against 2L of dialysis buffer (6-18hrs / 4°C).
Day 3
17) Titre by assaying serial dilutions cf comercial Taq.
18) Aliquot concentrated Taq polymerase and store at -20°C.
Reagents
Terrific broth (TB); per litre
12gm tryptone
24gm yeast extract
4ml glycerol (autoclaved).
100ml 0.17M KH2PO4(2.31gm/100ml) / 0.72M K2HPO4 (12.54gm/100ml);
Autoclaved separately.
Buffer A (Require 100ml)
50 mM Tris (pH 7.9)
1mM EDTA
50mM Dextrose
Buffer B / 100ml (Require 1000ml)
20mM Hepes (pH7.9) 2ml (1M)
1mM EDTA 1ml (0.1M)
0.5% Tween-20 0.5ml
0.5%NP-40 0.5ml
0.5mM PMSF
50mM KCl
Buffer C / 500ml (Require 500ml)
20mM Hepes (pH 7.9) 10ml (1M)
1mM EDTA 5ml (0.1M)
0.5% Tween-20 2.5ml
0.5%NP-40 2.5ml
0.5mM PMSF
200mM KCl
Dialysis Buffer / 2L (Require 2000ml)
20mM Hepes (pH 7.9) 40ml (1M)
1mM EDTA 4ml (0.5M)
0.5mM PMSF
100mM KCl
50% glycerol 1L
1mM DTT
Dilution Buffer Required to dilute Taq
20mM HEPES (pH 7.9)
0.1mM EDTA
100mM KCl
50% glycerol
Keywords: Taq, PCR, polymerase
Contact Email Address: Ian.Sp...@nott.ac.uk
Submitted at 17:27 on 22/1/96 by:
Dr. Ian Spendlove,
Academic Clinical Oncology,
City Hospital,
Huknall Road
Nottingham,
NG9 1PB,
U.K..
Tel: +44 (0)115 9691169 ex47608
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Though those buffers might be a bit of a show stopper for "home-made",
hopefully all of the chemical suppliers will be nice and friendly
about it..
- Bryan