yesterday night current went off, i slept i don't know when current came back. why r u telling no?
On Friday 2 March 2012 at 3:54 AM, diybio@googlegroups.com wrote:
Group: http://groups.google.com/group/diybio/topics
- Offtopic: New Google Groups [1 Update]
- Which microscope? [7 Updates]
- ultracentrifuge [7 Updates]
- Myostatin inhibitors again [3 Updates]
- Community Lab in Melbourne, Australia [3 Updates]
- [Open-rtms-list] tinkering [1 Update]
- Designer Probiotics. Anyone interested in this? [2 Updates]
- Question on DNA length detection via electrical methods [1 Update]
Cory Geesaman <cory@geesaman.com> Mar 01 02:07PM -0800
The new Google groups look cool, guessing not many have logged in to see it
yet since there's only 3 +1's at the top. Worth checking out.
Mega <masterstorm123@gmail.com> Mar 01 02:27AM -0800
I asked the proffessor if I could have access to the lab to do a
transformation. And guess what.
Monday I'm doing it.
But there are still a few questions...
When I make an LB Amp Agar Dish, I first mix water and LB Agar. Then I
heat it up. I think making 50 ml will be enough for one or two petri
dish.
Then I have to sterilize it. Will 3 mins in the microwave do the job,
or will I need to put it into the autoclaveur for 20mins as
recommended?
And, at <55°C I have to add the ampicillin which is not heat
ressistant.
Mega <masterstorm123@gmail.com> Mar 01 02:29AM -0800
Oh, just recognized it was renamed. Shall I start a new discussion
thread?
Cathal Garvey <cathalgarvey@gmail.com> Mar 01 10:49AM
Hey, awesome to hear that you're covered for a location! That's a really
helpful Prof you've got there. See, academic spirit isn't dead yet!
For your time-sensitive experiment, don't risk a failed sterilisation on
a microwave you haven't used previously for this task. I'm enthusiastic
about microwaves being an effective sterilisation tool, but I'm still
not confident enough of their reliability to recommend them when an
autoclave is at hand!
So, autoclave, then wait until it's cooled enough that you can handle it
without pain yet it's still liquid. That's a good time to add Amp; do it
under sterile conditions, such as close to a blue bunsen flame or under
a HEPA-filtered airflow. Don't shake to mix; cooled molten agar forms
very stable bubbles! Swirl patiently to mix. You're aiming for 100ug/ml
Ampicillin, so for 50mls you want 5mg of Amp.
It's normal to make stock solutions of ampicillin at 1000x (100mg/ml)
strength, and filter sterilise them, but if you can't afford the filters
you'll get away without sterilising the Ampicillin stock. After all,
it's a strong antibiotic! Just don't expect the resulting agar plates to
last forever if you can't sterilise the Amp; make them up fresh and use
within a week or two.
On 01/03/12 10:27, Mega wrote:
--
www.indiebiotech.com
twitter.com/onetruecathal
joindiaspora.com/u/cathalgarvey
PGP Public Key: http://bit.ly/CathalGKey
Mega <masterstorm123@gmail.com> Mar 01 04:20AM -0800
Well, so what if I diluted 5mg Amp in 10 ml water?
LB-Agar powder for 50 ml in 40 ml water, afterwards you add 10 ml amp-
ater and you've got 50 ml lb-agar+amp water ??
>Swirl patiently to mix.
When I can touch the Petri dish with my hands without pain, I'll add
the amp. How long will the agar still be liquid to swirl it?
Nathan McCorkle <nmz787@gmail.com> Mar 01 10:53AM -0500
You're going to need LB liquid too for the overnight and 3-5 hour culture
prior to transformation, as well as 2ml for the post-transformation
recovery period... so make about 150ml total LB, split into three 100ml
flasks each with 50ml in (autoclave fluid at half capacity of container).
Add the agar to one of the three flasks before autoclaving, then after
autoclaving when its cool enough that the flask is comfortable to hold with
your bare hand, add the ampicillin to the agar flask only, and mix to
swirl, then pour into the petri dishes (25ml per dish should work).
50ml LB with a scrape of e.coli culture and incubate in shakers incubator
overnight, or about 12-18 hours.... Then add about 5 ml of that in 30ml of
fresh LB and incubate in shaker for 3-5 hours just prior to transformation.
You'll need a centrifuge if you want to do CaCl2 method, basically just
take 1ml of the 3-5 hour culture, and centrifuge it in a microtube, the
cells collect at the bottom, and dump off the LB media, replace with 1ml
CaCl2, vortex to resuspend, centrifuge again, dump off liquid, resuspend in
fresh CaCl2, repeat maybe one more time and then let chill on ice... Add
DNA, chill, heat shock, add 2ml fresh LB, incubate in shaker for 1 hour,
plate on LB agar with ampicillin... (This is the basic idea, protocols may
include additional chilling steps, etc). Read the manual I posted from my
school.
Mega <masterstorm123@gmail.com> Mar 01 10:58AM -0800
reg. the plates:
http://www.benchfly.com/video/164/how-to-pour-lbagar-plates/
This video was very helpful (second vid)
Thank you for your help!!
Well, I could either dose the LB medium as it is said on the LB-Agar
package:
10g LB-Agar per liter. I don't heat it, so the agar will sink to the
ground. The supernatant is then LB-water.
Or I just use 'a lot' of LB-Agar (without calculating) and add some
water. What will stay at the ground of the tube is then agar mixed
with undissolved LB. (Not efficient, but for very small dosages maybe
ok)
Nathan McCorkle <nmz787@gmail.com> Mar 01 04:04PM -0500
> water. What will stay at the ground of the tube is then agar mixed
> with undissolved LB. (Not efficient, but for very small dosages maybe
> ok)
Huh?
You need to heat agar, or it won't dissolve and would be pointless to add.
Don't use too much or the salt concentration will be wrong, why wouldnt you
just use what the package specifies?
Or, are you saying you don't have LB without agar already added?
> --
> You received this message because you are subscribed to the Google Groups
"DIYbio" group.
> To post to this group, send email to diybio@googlegroups.com.
> To unsubscribe from this group, send email to
diybio+unsubscribe@googlegroups.com.
> For more options, visit this group at
http://groups.google.com/group/diybio?hl=en.
Phil <philgoetz@gmail.com> Mar 01 09:03AM -0800
> 200,000g would probably mean Ti rotors and an armour plate centrifuge chamber. If you really want that scale of speed and can manage with small volumes ie ~100uL consider using compressed air instead (a la Beckman airfuge). Only one moving part in the fuge, and you can hit 500,000g. Still going to need a Ti rotor and armour plate though...
I agree entirely. You can pick up a used Airfuge on ebay for about
$50. You'll have to buy an aluminum rotor for it, for about $1800,
from Beckman. Do not try to build your own ultracentrifuge rotor.
You will probably want to run the Airfuge inside a refrigerator
anyway, and that will provide some protection in case of disaster.
Jeswin <phillyj101@gmail.com> Mar 01 12:19PM -0500
> You will probably want to run the Airfuge inside a refrigerator
> anyway, and that will provide some protection in case of disaster.
How about in a concrete filled washing machine?
John Griessen <john@industromatic.com> Mar 01 11:47AM -0600
On 03/01/2012 11:03 AM, Phil wrote:
> Do not try to build your own ultracentrifuge rotor.
Why make such statements on a DIY list?
John Griessen <john@industromatic.com> Mar 01 11:49AM -0600
On 03/01/2012 11:19 AM, Jeswin wrote:
> You will probably want to run the Airfuge inside a refrigerator
It might be its own refrigerator by expansion cooling.
If not, putting a flow orifice a little upstream of it would do some
refrigeration -- probably so much you'd need to control it to avoid freezing.
Cathal Garvey <cathalgarvey@gmail.com> Mar 01 06:06PM
Because an ultracentrifuge can kill you and is highly prone to explosion if used without training?
--
Sent from K-9 Mail on Android
John Griessen <john@industromatic.com> Mar 01 01:59PM -0600
On 03/01/2012 12:06 PM, Cathal Garvey wrote:
> Because an ultracentrifuge can kill you and is highly prone to explosion if used without training?
Isn't this list about training? I see it going on all the time. We have someone
researching ultracentrifugation and why not evolve a design? Not me saying
"Do it this way and you'll be guaranteed safe.", but what guarantees are there
with anything DIY?
So, we've had comments about concrete for mitigating centrifuge risk, and that's
good. Concrete is good. Steel plate is good. They go together well.
Concrete is easy to form around any plastic as a mold that releases easily.
So, a containment well with tapering narrower as it goes up top can easily be made
for the motor/rotor to fit in with a 1/2 in steel lid over it. All the kinetic energy
of the ultra rotor is angular and won't change easily, so forces are sideways and gravity
keeps it down. Precession can change the angle a little, so sloping inward sides
are good to keep it down, not let it climb even if it starts precessing around the
containment well interior. Lining the cast concrete interior with smooth plastic
such as HDPE would be great for keeping the concrete surface from crumbling by an
attacking loose rotor -- how would you hot spray coat that plastic?
Let's talk about it, OK?
Make your ultra rotor 6 cm across of Ti metal on an air bearing, driven by
an air turbine. Let it hold vials with a cone bottom that are 2 cm deep and 1 cm across
holding maybe 1.2 ml at the full mark. What turbine parts are out there that can be re-purposed?
What bearings does the Portescap motor company recommend for their 70,000 RPM motors?
Why not talk about it?
Cathal Garvey <cathalgarvey@gmail.com> Mar 01 08:21PM
I'm all for informed risk, just saying why people suggest the safe alternative! :)
--
Sent from K-9 Mail on Android
Bryan Bishop <kanzure@gmail.com> Mar 01 10:32AM -0600
Here's a project proposal from yashgaroth.
-----------------------------------
by *Yashgaroth<http://transhumani.com/member58.html?sid=a31e04e010769bbe4beabedf8e55223b>
* » Wed Feb 29, 2012 10:58 pm
Out of the many possibilities in DIY gene modding, myostatin inhibition to
augment muscle growth is the most immediately doable. For a thorough and
current review on the topic, I highly recommend S.J. Lee's recent paper,
available at http://www.jhu.edu/sejinlee/downloads/Lee%20IEMAMC%202010.pdf .
If you don't feel like reading that first, here's a brief overview:
myostatin is a protein which, in skeletal muscle, is part of the network
that negatively regulates muscle mass. By inhibiting myostatin and/or its
related proteins, up to a quadrupling of muscle mass has been observed in
transgenic mice, with naturally inhibiting mutations in the gene leading to
the "double-muscled" phenotype found in certain breeds of dogs, cattle, and
that one kid in Germany you may have read about.
My hope is to express a protein called follistatin in muscle tissue, as
follistatin is a highly potent inhibitor of myostatin. Currently,
follistatin is receiving much interest for treating muscular dystrophy; a
clinical trial starts this month, based on research that culminated in this
paper:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2852878/ . Basically,
they're doing what I plan to do, although they will be using a viral vector.
Since viral vectors require a great deal of expense to produce, as well as
a regimen of immuno-suppressive drugs, they don't really work for the DIY
approach right now. Among the non-viral vectors, plasmid electrotransfer
(or electroporation) is the most powerful, and, as a bonus, it requires no
reagents other than electricity.
The plasmid backbone is fairly simple: standard CMV promoter, synthetic 5'
intron, SV40 polyA and enhancer, all to increase expression of the
follistatin transgene as much as possible. Beyond that, I'm thinking a
couple of short S/MAR insulators flanking the antibiotic resistance gene,
which should be CpG-depleted to prevent chromatin condensation.
Anyway, I'm not too good at writing long and coherent statements, so the
rest of this is some of my notes detailing the execution of the project. I
welcome any comments and critiques.
On the topic of which gene to express, a few alternatives to follistatin do
exist:
- Anti-myostatin monoclonal antibody (developed by Wyeth), perhaps the
simplest method, from the old "raise an antibody against it" school of
thought. Made it to clinical trials, but was not pursued as it caused skin
rashes.
- Fusion of a mutant myostatin propeptide to an antibody Fc region
(fusion to increase the half-life at least 100-fold). The propeptide
normally gets produced with myostatin, and blocks its acivity until cleaved
at a later point, which "activates" the mature myostatin; the mutant
propeptide is resistant to said cleavage, and sequesters myostatin. Showed
some efficacy in a canine model, but has not yet been pursued.
- ACVR2B solubilized receptor (Acceleron Pharma and Amgen), again fused
to Fc for half-life extension like the propeptide above. ACVR2B is the
"main" receptor for myostatin in muscle, so the soluble extracellular
receptor was intended to soak up circulating myostatin, similar to the
previous two approaches. This worked incredibly well in mice, but triggered
an increase in hematocrit in human trials (due to its non-muscle effects),
so it got shelved.
All of these options were pursued by injecting recombinant proteins into
the bloodstream, rather than a gene therapy approach. This means that
off-target effects on organs other than muscle must be carefully
controlled. With gene therapy, the transgene can be targeted to a specific
location, where expression will occur along the length of the muscle fiber,
but not spread to the heart, skin, gonads, etc. This also opens up a number
of other possibilities, including intracellular protein expression or RNA
interference, which would not be possible with a protein-injection-based
therapy. For example:
The ACVR2B soluble receptor comes a close second to follistatin as a choice
of transgene. One simply removes the intracellular kinase domain, with
which the receptor would normally transmit the myostatin signal. The
extracellular myostatin-binding domain is all that remains, which then
soaks up myostatin. Note that this would retain the transmembrane anchoring
region, safely limiting it to the injected muscle. Unlike the Fc fusion
protein mentioned above, this should not affect RBC count.
Please note that I say 'myostatin' here, but ACVR2B and follistatin appear
to bind additional muscle-limiting ligands beyond myostatin.
Myostatin-knockout mice still exhibit increased muscle growth with
administration of either transgene, indicating at least one other factor at
play.
Plasmid production:
Plasmids can be grown in a standard e. coli culture, and purified either
with the commercial endofree giga-prep kits (Qiagen or a cheaper
competitor) or, with some investment but a lower $/mg cost, column
chromatography: standard alkaline lysis protocols, followed by a
Triton-X114 precipitation (to remove endotoxin), and a DEAE + hydrophobic
interaction column setup should give acceptable purity of plasmid for
injection.
Electroporator:
The parameters for large mammals haven't really been nailed down yet, so
I'd like one with variable settings that cover the range of the previous
experimental techniques. This would be a unipolar square-wave generator
capable of 20-200 volts, 0.1-0.5 amps, with pulses of 10-50 milliseconds in
duration, with a variable number and spacing of said pulses. Optimal
settings for humans are hard to pin down due to the difficult in assaying a
reporter gene in humans, so I am aiming for the maximum tolerable settings
that do not produce unnecessary tissue damage.
My favorite applicator so far is a carriage enclosing two 1mL needles,
which simultaneously inserts the needles while depressing the plunger,
ensuring an even distribution of plasmid along the length of the needle.
Once injection is complete, the needles act as the electrodes.
The only other modern technique in practice involves multiple electrodes
positioned around an injection needle, which then fire in different
combinations. This is seen in most clinically available electroporation
devices, and while I prefer the two-needle technique, the pulse generator
should be easily adaptable to an arbitrary arrangement of needles.
-----------------------------------
http://transhumani.com/topic61.html
- Bryan
http://heybryan.org/
1 512 203 0507
irc.freenode.net ##hplusroadmap
Nathan McCorkle <nmz787@gmail.com> Mar 01 12:30PM -0500
How would the plasmid target muscle cells only, not including the heart?
Mouse liver glowed when intranasal plasmid was administered, so
electroporation may not be necessary
mad_casual <ademlookes@gmail.com> Mar 01 10:43AM -0800
> How would the plasmid target muscle cells only, not including the heart?
Myostatin and MyoD are skeletal muscle genes and electroporation is
local. IMO, the concern is the other way; non-genetic DIYBio muscle
growth has been going on for 60 yrs., there's plenty of non-clinical
evidence to suggest that rapid growth of muscle and decades of walking
around with tons of extra muscle (pharmacologically induced or not)
doesn't do your heart or connective tissues any favors. If I were 20
yrs. old, 6'2" and bulletproof, I'd probably try this, once my kids
are out of the house and I'm having trouble keeping up in the gym, I
might try something like this. Right now, it just seems like a bad
idea.
Christopher Pendlebury <mailinglists@christopherpendlebury.com> Mar 01 07:47AM -0800
Hi everyone,
I'm embarking on a project to build a community lab in Melbourne,
Australia, with a focus on biology, biotechnology and bioinformatics.
Because of some of my contacts, the bioinformatics aspect could
potentially include high-end NGS analytics.
The lab will be bootstrapped, and funded by memberships and
sponsorships. I would very much like to know who would be interested
in being involved with this project, and who would like to join when
it is up and running?
Cheers
Chris
Avery louie <inactive.e@gmail.com> Mar 01 01:18PM -0500
I am in the US, but good luck! We need more people like you.
--Avery
On Thu, Mar 1, 2012 at 10:47 AM, Christopher Pendlebury <
Sumon Sadhu <sumon.sadhu@gmail.com> Mar 01 10:25AM -0800
It would be really interesting to understand the costs of building a
community lab, and how cheap or expensive things are, what equipment you
intend to have etc. Care to share your thinking here? I think it could
spark a discussion around how it could be done in more places round the
world.
Bryan Bishop <kanzure@gmail.com> Mar 01 10:30AM -0600
From: Tobias Schäfer <tobias.schaefer@tu-clausthal.de>
Date: Thu, Mar 1, 2012 at 4:37 AM
Subject: Re: [Open-rtms-list] tinkering
To: open-rtms-list@lists.sourceforge.net
Hi!
Yes, there is hardware:
http://open-rtms.sourceforge.net/roadmap.html
On the bottom of that site is the section Electronics
In the Gallery You can find a photo of the hardware I put together.
Status of the project:
Parallax Propeller:
The objects for keyboard, mouse, VGA and communication are provided by
the manufacturers.
I have written objects for graphics/text overlay and for sending out
signals on 8 channels at precisely the same time.
What is missing is the generator object for "complex signals".
Basically: What is meant by "complex signals"?
Nice-to-have would be a object to measure the resistance R and the
inductance L of a connected coil.
Control software:
I have written a class for connecting to the Propellers build-in
bootloader and uploading a program to it (and writing it to the EEPROM
also).
Works for Linux. For Windows the control of the DTR-line is missing in
the SerialPort class. This line is needed to reset the Propeller to get
it into the bootloader. I will check that out, if I start the
cross-compiling experiments.
I have written an FEM solver for magnetic fields around coils. I want to
use it to calculate the fields inside the brain.
I am also porting & integrating an old (2005) program of mine to display
tomography data. (A friend of mine got a scan of his brain on CD, when
he volunteered in some medical experiments at his university. One of
those if-I-poke-you-here-what-part-of-your-brain-gets-activated
experiments.)
Missing:
The glue in between. What is the workflow of the program? Where do I
click? What happens then?
For tDCS: I assume You want to imprint currents in the area of around 1
uA. My crude measurement of the resistance temple to temple gives around
1 MOhm. So simply take the electronics and change Rmeas to 1 MOhm. Now
the circuit should be able to imprint currents of -2.5 uA to 2.5 uA.
Possibly a little thought has to be spend on the OP-Amp selection and it
might get tricky in a multi channel setup to get all the relative
voltages right.
Am 01.03.2012 05:17, wrote James Braddock:
> I can fudge the software in place good enough, but hardware I'm no good.
> Thanks
> jb
------------------------------------------------------------------------------
Virtualization & Cloud Management Using Capacity Planning
Cloud computing makes use of virtualization - but cloud computing
also focuses on allowing computing to be delivered as a service.
http://www.accelacomm.com/jaw/sfnl/114/51521223/
_______________________________________________
Open-rtms-list mailing list
Open-rtms-list@lists.sourceforge.net
https://lists.sourceforge.net/lists/listinfo/open-rtms-list
--
- Bryan
http://heybryan.org/
1 512 203 0507
CodonAUG <elsbernd@gmail.com> Mar 01 06:22AM -0800
There is no 'business of skeptical criticisms of science' because
skeptical criticism is the the norm for science.
Richard Proctor <richardmproctor@gmail.com> Mar 01 07:09AM -0800
I doubt that the side effects of diaorrhea would be a problem with
people using this potential probiotic so long as they are made aware
of it.
lets not forget that one of the most widly used obeesity medications
orlistat comes with the side effect of squits if you eat to much fat.
Infact they have made it a selling point of providing an " adversion
therapy."
I think it is absolutely necceseary to build in some kind of adversion
into an anti obesity treatment. You don't want to send the message
that " woohay science lets you eat as much crap as you like and never
get fat." All that will lead to is an increse in US levels of
consumption.
Pat <eleten@gmail.com> Mar 01 05:13AM -0800
If you were able to isolate the strand in question you could probably
run it through something very similar to the nano pore technology in
the new sequencers. Instead of reading the electrical signature and
correlating it with the chemical base you would just read from the
first electrical signal to the last.
http://www.gizmag.com/minion-disposable-dna-sequencer/21513/
You received this message because you are subscribed to the Google Group diybio.
--
You can post via email.
To unsubscribe from this group, send an empty message.
For more options, visit this group.
You received this message because you are subscribed to the Google Groups "DIYbio" group.
To post to this group, send email to diybio@googlegroups.com.
To unsubscribe from this group, send email to diybio+unsubscribe@googlegroups.com.
For more options, visit this group at http://groups.google.com/group/diybio?hl=en.
You received this message because you are subscribed to the Google Groups "DIYbio" group.
To post to this group, send email to diybio@googlegroups.com.
To unsubscribe from this group, send email to diybio+unsubscribe@googlegroups.com.
For more options, visit this group at http://groups.google.com/group/diybio?hl=en.
0 comments:
Post a Comment