Science

I am not familiar with your technique, do you have any more details
about it? Maybe I can offer some ideas.

On Jan 20, 2:26 am, David McWilliam <dmcwill...@gmail.com> wrote:
> Great post. I've been looking for something like this for macerating plant
> material in bead beater tubes prior to DNA extraction. I've just  bought a
> dremelfuge which arrived today and am thinking of ways to attach it to a
> vortexer. The horizontal tube position would help maximise the bead
> vortexing although it may need something to stop the tube caps from
> popping. Maybe a vortexer modification of the dremelfuge - Cathal?
> DMcW
>
> On 20 January 2012 15:59, CodonAUG <elsbe...@gmail.com> wrote:
>
>
>
>
>
>
>
> > I wrote up a blog post about how to make a tube holder for vortex
> > mixers.  I needed something to hold microcentrifuge tubes for me
> > because I didn't want to stand there for 10 minutes with my arms
> > getting uncomfortably shaken.
> > Check it out if you are interested.
>
> >http://cheapassscience.wordpress.com/2012/01/20/diy-tube-holder-for-v...
>
> > --
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In response to Jonathan's initial question, you're not wrong - the
primers will denature before 94, but the full-length PCR product
you're trying to amplify is a lot longer and requires a higher melting
temp to dissociate.

also, if you're designing primers you can find some convenient code at
https://github.com/russelldurrett/Python_Primers

On Jan 27, 11:43 am, Jonathan Nesser <jonathan.nes...@gmail.com>
wrote:
> Sorry to start yet another new thread for such a small question, but I
> don't want to pollute other threads with off topic posts... I've been
> trying to understand why primer melting temperatures have to be in the
> 55-65C range when PCR cycles call for 94C for DNA melting... I've tried to
> find an explanation for this but haven't been able to... Wouldn't that
> result in the primers splitting off from the template DNA before you reach
> the extension cycle of 75C (or around there)? I'm sure I'm wrong because
> obviously these PCR programs work, but I don't know WHY I'm wrong, and
> would like to understand. Thanks again for explaining something that is
> probably so common knowledge that nobody feels the need to address it in
> writing :)
>
> Jonathan Nesser
> jonathan.nes...@gmail.com
> diybioandneurosci.blogspot.com

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Thank you very much Cathal! This is great - matches up with what I've
found in my research (I really didn't want to have to culture, and PCR
is more sensitive anyway). Thanks in particular for the tips on
boiling and mixing with alcohol.

Now, are you suggesting I just run gels at home? This is certainly an
option, but I get the feeling this will be pretty labor (and reagent)
intensive compared to relying on a lab equipped with the right
automation (especially because for each sample I might need to test it
against >10 different viruses). Also I'd love to get some indication
of viral load if possible. Multiplexed qPCR seems to be an emerging
standard here, but I'm not sure how to find a lab I can buy services
from on samples I send (and, at first glance at least, a qPCR machine
is probably still too expensive for me to buy myself). As an example,
here's a couple of the promising products I've found for multiplexed
qPCR respiratory virus panels:
http://www.luminexcorp.com/Products/Assays/ClinicalDiagnostics/xTAGRVP/index.htm
http://www.idahotech.com/FilmArray/RespiratoryTest.html

Another option (if I give up on measuring viral load) is Sanger
sequencing - at least there are lots of cheap services advertised
here. Perhaps with carefully chosen primers I could identify the most
common respiratory viruses with ~10 reactions (which looks like I
should be able to get for <$50/sample). Getting a bit of sequence
would be a nice bonus for eventually tracking specific viral strains.
Is this a viable option worth considering do you think?

Thanks!
Rick

On Jan 31, 10:35 am, Cathal Garvey <cathalgar...@gmail.com> wrote:
> Hey Rick,
> I think PCR is almost certainly the way to go, for a few reasons.
>
> Firstly, you're talking about mammalian viruses; to grow them (which is
> a bad idea in any case), you'd need mammalian cells, and mammalian cells
> call for more intensive care than bacteria. It's a big investment, and
> it requires a lot of time and effort just to keep them going.
>
> And that's ignoring the fact that each virus will call for a different
> target cell type, which in turn calls for different care procedures;
> kidney cells may need very different medium and care than liver cells,
> for example.
>
> Secondly, plaque assays don't really work, AFAIK, for mammalian viruses.
> Because they must be grown with a liquid layer on top, and that liquid
> layer allows viruses to spread freely, instead of growing outwards like
> bacteriophages.
>
> Besides, if dealing with human infections, a good rule #1 is *never
> culture*. If you can do your work without culturing the viruses at all,
> then you should.
>
> DNA survives boiling and alcohol, but viruses don't. If you want to
> study your kids' colds, you could just take a sample of nasal mucous,
> boil it in an eppendorf tube for 10 minutes, add 40% EtOH just to be
> sure and then use a tiny sample for PCR, using primers that will amplify
> your virus of interest. Ideally use primers that are already tested and
> shown to work reliably in the available science literature. If you make
> your own, aim for 25 nucleotides in length in a non-repetitive area of
> the virus genome that's probably well-conserved, such as promoters.
>
> PCR is great for many reasons. One reason is that it allows you to study
> something that is impossible or unwise to culture, such as thermophiles,
> bizzarre soil symbiotes, or human pathogens. I would strongly suggest
> sticking with PCR and taking normal precautions to avoid infecting
> yourself while you take samples.
>
> On 25/01/12 05:51, Rick Byers wrote:
>
>
>
>
>
>
>
>
>
> > Hi,
> > I'm curious to better understand the pattern of viral infections in my
> > family (I've got two young kids, so it's pretty common for some cold
> > to be going around our house).  I'm trying to figure out the most
> > practical way to do regular URT diagnostics.  I've got no lab
> > experience (but would love to learn) and almost no lab supplies (but
> > am willing to invest up to a few thousand dollars as necessary).
> > Ideally I'd like something that is both quantitative (so I can track
> > viral load over the course of infection) and multiplexed (so I can
> > identify which viruses are responsible and catch most common ones).
>
> > I know I can do some simple diagnostics at home with a modest
> > investment.  Eg., I considered doing simple plaque assays or simple
> > PCR runs, but I'm hoping I can find something that's less time
> > consuming at non-trivial scale (eg. detecting at least 10 different
> > viruses/strains, ideally many samples over a period of time).  Perhaps
> > there's an immunoassay panel of some sort I could use at home?  Or
> > perhaps the best option is a service that will do a multiplexed qPCR
> > run for me on a set of samples I send in.
>
> > I'm continuing to do research of various products and diagnostics
> > services, but I'm finding it pretty difficult to get specific prices
> > and weigh the options.  Any advice would be greatly appreciated.
> > Thanks!
>
> --www.indiebiotech.com
> twitter.com/onetruecathal
> joindiaspora.com/u/cathalgarvey
> PGP Public Key:http://bit.ly/CathalGKey

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That can't be printed on an FDM printer like a makerbot because of the overhangs. It'd either need to be redesigned, broken up, printed with supports, or done with a different process like SLS.

Alternately, the idea looks ideal for adaptation to a laser cutter.

First, Chemists didnt limit the supply of chemicals the DEA/FBI did and they didn't just limit chemicals, they also restricted glassware , at least here in Texas.

On 01/31/2012 02:08 PM, kingjacob wrote:
> But this is relevant ;) http://citizensciencequarterly.com/2010/11/26/swiss-army-tube-block/

Looks like a difficult size and volume of plastic to 3DP with a makerbot
or such, at their current state of capability -- a void or gap would ruin
a long print run, and the qty of plastic is large for those plastic filament
printers.

It IS a great design though, for when 3DP becomes cheap/quick.

Meanwhile a drill press and table or radial saw and salvaged
chunks of hardwood or plastic and you have similar function.

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I just use the boxes they send enzymes in.

cigar box with sand in it

But just because it is not a good idea, somebody is going to try it if
tech becomes easier and cheaper. Prevention is nice, but strictly
followed has to kill every DIYBio idea. Chemists have limited the
supply of chemicals, but bombs are build anyway. What about making a
lot of biobricks easy available but somehow limit/control the service
of synthesis companies/machines?

On 24 Jan., 11:27, Cathal Garvey <cathalgar...@gmail.com> wrote:
> I'd have said that the main problem combining DIYbio and vaccines was
> the risk of creating a novel, dangerously infectious virus if someone's
> idea of "attenuated" isn't quite up to scratch!
> This was a theme at the DIYbio London Congress where we threshed out a
> "DIYbio Code": Really, working with infectious organisms at home is a
> seriously bad idea, and shouldn't be allowed to share the "DIYbio"
> umbrella-title, lest we all suffer massive regulatory backlash for
> someone's overambitious mistakes.
>
> Leading to my personal opinion; making medicines at home is pretty OK
> provided they aren't extremely toxic, but making artificially attenuated
> vaccines is dangerous business. A home lab is *always* prone to the
> kid-next-door barging in, and Chekhov's law will always win out; if
> there's a gun on the wall, it'll be fired before the play's over. :)
>
> On 24/01/12 09:16, Pieter wrote:
>
>
>
>
>
>
>
>
>
> > Great to hear that there will be a synbio conference again in The
> > Netherlands. The DIYbio movement here could definately use some
> > support. So if you are able to break any ground for us, just let me
> > know.
>
> > There are quite a few DIY Bio projects going on involving healthcare,
> > but as far as I know they do not incorporate the SynBio approach.
> > Problem with combining DIY Bio and medicine is the validation part.
> > Although it might sound relatively easy to produce antigens in cell
> > lines, almost the entire flu vaccine industry still runs on the same
> > old chicken egg method that was validated decades ago. In Holland
> > there is a MDCK cell line vaccine facility in Weesp, but it is
> > relatively small in size. In such a competitive industry as flu
> > vaccines, it is hardly worthwhile to invest in these kind of new
> > production methods.
>
> > On Jan 24, 3:05 am, Thomas Landrain <thomas.landr...@gmail.com> wrote:
> >> Hey Cathal!
>
> >> Thanks a lot for your input, I am definitely going to use it. Very cool project BTW. Your work is paving the amateur's roads to SynBio, and that's what counts!
> >> I'll definitely share the finalized document with community, it should come shortly in fact, the deadline is next Wednesday ;)
> >> Cheers!
>
> >> Thomas
>
> >> PS: You're more than welcome in Paris! Actually, we should maybe start to think of a "DIYbio Europe Congress 2012", Paris could host that! ;)
>
> >> On Jan 23, 2012, at 12:37 PM, Cathal Garvey wrote:
>
> >>> Hey Thomas!
> >>> Delighted to hear La Pailasse is growing so successfully. Can't wait for
> >>> my next excuse to visit Paris!
>
> >>> My own projects at this very moment have no especial significance to
> >>> medicine, but I'd like to leverage them if they succeed into the realm
> >>> of decentralised medication production.
>
> >>> Specifically, I'm still working on my synthetic Bacillus plasmid. Chief
> >>> barriers until now have included a lack of positive control plasmids for
> >>> Bacilli, which I've now sorted out, and a few outstanding paperwork jobs
> >>> I need to do for legal reasons before starting.
>
> >>> If the plasmid works, I'll be offering it for sale as open-source DNA
> >>> for DIY Synthetic Biology; it's biobrick compatible and hopefully it
> >>> will not need antibiotic selection to work.
>
> >>> What *I'd* like to do with it first though is to immediately attempt
> >>> producing a useful medicine; thyroxine, for example, or an antibiotic. I
> >>> wrote at modest length about the potential for production of thyroxine
> >>> using Bacillus subtilis on my blog:
> >>>http://www.indiebiotech.com/?p=135
>
> >>> The prices of attempting this have since fallen significantly provided
> >>> one is willing to do a little in-lab assembly, as IDT are now offering
> >>> 500bp blocks of DNA for $99 each; cost per nucleotide drops to $0.20/bp
> >>> at that rate, although allowing for overlaps for assembly the effective
> >>> cost-per-bp is slightly higher.
>
> >>> Vaccines are something that synthetic biology has *tremendous* power to
> >>> deliver in short order, but it's not an area that would be prudent or
> >>> safe for DIYbio-scale engineering. Far better to cut teeth at the DIYbio
> >>> level, then found or work within an existing BL3/4 lab to do work with
> >>> more dangerous systems.
>
> >>> Still, although it's not "DIYbio", the production of a new flu vaccine
> >>> could be rapidly accomplished through synthetic biology by creating
> >>> complementary strains of host cell lines in which to grow artificially
> >>> crippled viruses. A cell line that produces viruses containing "almost
> >>> dud" genomes, just enough to stimulate infected cells to produce and
> >>> display viral proteins via MHC, would stimulate every natural level of
> >>> immunity, both innate and adaptive, without any reasonable risk of
> >>> back-mutation. That we're not already producing vaccines in this way is
> >>> pretty headache-inducing to me.
>
> >>> Finally, the "medical" benefits of access to lifestyle-altering drugs
> >>> such as contraceptives shouldn't be overlooked, either. If strains of
> >>> bacteria can be coaxed to make (non-environmentally persistent) forms of
> >>> oestrogen or progesterone that could be used for contraception, the
> >>> provision of cheap and reliable synbio contraceptives could go a long
> >>> way towards improving global healthcare.
>
> >>> And that's before you start discussing symbiotic, sub-dermal cultures of
> >>> hormone-producing bacteria; freckles that help keep you infertile until
> >>> you want to conceive!
>
> >>> Looking forward to the report from La Paillasse. Keep up the awesome work!
>
> >>> On 22/01/12 00:45, Thomas Landrain wrote:
> >>>> Hey DIYbio enthusiasts,
>
> >>>> While most of you don't know me, I will represent the DIYbio
> >>>> community for an international workshop organized by the SYBHEL
> >>>> project (European program) called "Synthetic Biology for Global
> >>>> Health: A policy discussion". It will be held in The Hague
> >>>> (Netherlands) on the 9th and 10th of February. Jason Bobe was
> >>>> originally invited but he asked me to replace him. For those that
> >>>> don't know me, my name is Thomas Landrain and I am co-founder and
> >>>> president of the first french community lab for biotech in France
> >>>> called "La Paillasse". Our french community is growing bigger at a
> >>>> constant pace, our microbiology/Molecular Biology Lab was installed
> >>>> few weeks ago and we are getting quite excited about the on-going
> >>>> projects we have. But, this is not my main job, everyday in fact I am
> >>>> designing new tools for Synthetic Biology based on RNA-RNA
> >>>> interactions as a PhD student. But I'll stop here for the
> >>>> presentation and will now focus on the real purpose of my email.
>
> >>>> Although I am not a regular contributor to this list, I'd like to
> >>>> request your help on gathering information concerning our broad
> >>>> community and the diverse realized/on-going/planned/wished realistic
> >>>> DIYbio projects, involving a Synthetic Biology approach, that could
> >>>> provide solutions to (global) health issues. I would like in fact to
> >>>> compile all those projects together into a document that would
> >>>> strengthen the positive perspectives of using SynBio in a
> >>>> non-institutional environment like DIYbio labs. You are all welcomed
> >>>> to contribute! Thanks in advance for your dear help!
>
> >>>> Thomas
>
> >>> --
> >>>www.indiebiotech.com
> >>> twitter.com/onetruecathal
> >>> joindiaspora.com/u/cathalgarvey
> >>> PGP Public Key:http://bit.ly/CathalGKey
>
> >>> --
> >>> 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.
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> >>> For more options, visit this group athttp://groups.google.com/group/diybio?hl=en.-Hide quoted text -
>
> >> - Show quoted text -
>
> --www.indiebiotech.com
> twitter.com/onetruecathal
> joindiaspora.com/u/cathalgarvey
> PGP Public Key:http://bit.ly/CathalGKey

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Quick note here

Annealing temp needs to be just low enough for primers to re-associate
with complementary strands. This is based on affinity, so, as a
previous poster pointed out, primer association % is in a state of
equilibrium.

Dissociation / Denaturing / Melting (whatever you want to call it)
tempurature is dependent on the bond strength between the template
strands, and the energy needed to dislodge Taq. The high (94 degree)
temp ensures that G-C (triple bond) rich regions are thoroughly
split. double-stranded DNA is super stable, so once the double helix
is formed (post elongation), you really need to blast the system with
heat energy before those bonds start to break.

On Jan 31, 10:36 am, Nathan McCorkle <nmz...@gmail.com> wrote:
> I think its wrong to say annealing only happens between 50-65 C, but
> that's the general range for most primers 15-25 bp in length
>
>
>
>
>
> On Fri, Jan 27, 2012 at 11:57 AM, veera <drveeramanikan...@gmail.com> wrote:
> > generally annealing temperatures will be just 3-4 C below Tm of primers. if
> > the melting point of primers is very high like you said in the range of 90 ,
> > at that hight tempertaure all double stranded dna will split off and so
> > annealing will not take place., because annealing happens only in the range
> > of 50 - 65 C. hope i'm correct.. if i'm wrong anyone can correct me...
>
> > Jonathan Nesser wrote:
>
> > Sorry to start yet another new thread for such a small question, but I
> > don't want to pollute other threads with off topic posts... I've been
> > trying to understand why primer melting temperatures have to be in the
> > 55-65C range when PCR cycles call for 94C for DNA melting... I've
> > tried to find an explanation for this but haven't been able to...
> > Wouldn't that result in the primers splitting off from the template
> > DNA before you reach the extension cycle of 75C (or around there)? I'm
> > sure I'm wrong because obviously these PCR programs work, but I don't
> > know WHY I'm wrong, and would like to understand. Thanks again for
> > explaining something that is probably so common knowledge that nobody
> > feels the need to address it in writing :)
>
> > Jonathan Nesser
> > jonathan.nes...@gmail.com <mailto:jonathan.nes...@gmail.com>
> > diybioandneurosci.blogspot.com <http://diybioandneurosci.blogspot.com>
>
> > --
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> > --
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> --
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> Rochester Institute of Technology
> College of Science, Biotechnology/Bioinformatics- Hide quoted text -
>
> - Show quoted text -

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"Once the size is large enough to hold 1 Atm of air pressure at
ground level you could feasibly start unlocking oxides in the soil to
make it livable"

Why that? Who says Mars can not hold 1 bar? Venus is Earth sized and
holds 90 bars!!
So Mars' Mass = 1/10 Venus' Mass => Atmosphere = 90/10 = 9 bar.

On 31 Jan., 17:40, mad_casual <ademloo...@gmail.com> wrote:
> Venus is a much more desirable candidate for many reasons:
> Mass- Mars isn't big enough to support an atmosphere fit for human
> habitation. You could float habitats on Venus' atmosphere.
> Productive Energy (thermal and non-ionizing radiation)- Mars' surface
> is relatively devoid of non-destructive energy. Venus has too much,
> even simple heat pumps would effectively convert atmospheric energy to
> other forms.
> Destructive Energy (ionizing radiation)- Mars is awash in it. Venus'
> atmosphere has a magnetosphere that affords a level of shielding.
> Chemistry- Mars has lower water, carbon, and nitrogen in any form on
> its surface or in its atmosphere than Earth. Venus has more of all of
> the above, typically boiled in acid and at 90 atm of pressure such
> that they are unusable to biological systems.
> Value- Fixing Venus teaches us lots about "fixing" Earth. Fixing Venus
> will be all about collecting energy rather than just expending it.
>
> IMO, put an outpost on the Moon, colonize Mars, terraform Venus.
> Anything else is making a purse out of a pig's ear. On that note; the
> human body is supremely adapted to this planet and for relatively
> short periods of time at that. Bending atmospheres to fit our lungs,
> putting chairs in spacecraft to fit our rear ends, and blasting
> surgeons and/or medicine around the Solar System is, to me, somewhere
> between naive fantasy and religion.

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Surely for a heat-pump to work, you need to have a gradient, not just
lots of heat.

To ask that another way: Are there larger heat *gradients* on Venus with
which to generate useful energy, or is there just more ambient energy,
which is of no use?

Venus is scary precisely because it used to be almost identical to
Earth, except that Earth developed life and Venus succumbed to runaway
global warming. There but for the grace of an ecosphere go we.

On 31/01/12 16:40, mad_casual wrote:
> Venus is a much more desirable candidate for many reasons:
> Mass- Mars isn't big enough to support an atmosphere fit for human
> habitation. You could float habitats on Venus' atmosphere.
> Productive Energy (thermal and non-ionizing radiation)- Mars' surface
> is relatively devoid of non-destructive energy. Venus has too much,
> even simple heat pumps would effectively convert atmospheric energy to
> other forms.
> Destructive Energy (ionizing radiation)- Mars is awash in it. Venus'
> atmosphere has a magnetosphere that affords a level of shielding.
> Chemistry- Mars has lower water, carbon, and nitrogen in any form on
> its surface or in its atmosphere than Earth. Venus has more of all of
> the above, typically boiled in acid and at 90 atm of pressure such
> that they are unusable to biological systems.
> Value- Fixing Venus teaches us lots about "fixing" Earth. Fixing Venus
> will be all about collecting energy rather than just expending it.
>
> IMO, put an outpost on the Moon, colonize Mars, terraform Venus.
> Anything else is making a purse out of a pig's ear. On that note; the
> human body is supremely adapted to this planet and for relatively
> short periods of time at that. Bending atmospheres to fit our lungs,
> putting chairs in spacecraft to fit our rear ends, and blasting
> surgeons and/or medicine around the Solar System is, to me, somewhere
> between naive fantasy and religion.
>

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The one in Ireland is my personal lab, but I'd be really happy to help
set up a community lab, also.

Getting a license to work with GMO bacteria was annoying and time
consuming in Ireland. My friends in Germany tell me it's next to
impossible for an individual, over there. It would have to be a
community lab, and there is a lot of legal risk even then.

I hope you get a group together who are willing to take that
responsibility and risk. The EU needs a big push to keep up with
small-science.

On 31/01/12 17:09, swish wrote:
> I don't think you are having much luck in Germany. There is one in
> Ireland.
> If you are interested in starting one we can maybe work together. I
> would like to start one Frankfurt (or Hessen if it is to expensive
> there). Our Idea was to go for S1 Labs at Schools at first. Maybe we
> could work out a concept to start one next year (but this year is
> really busy) It would be an advantage to have several Labs and a
> community in Germany to deal with legal stuff.
>
> Greets
> Sebastian
>
> On 26 Jan., 19:39, Felix Maier <ukai....@googlemail.com> wrote:
>> Are there any DIYbio labs in Berlin, Germany to join ?
>> Anyone building up a DIYbio lab for i.e. workshops?
>> I am very interested in this subject.
>>
>> Feel free to contact me via email.
>> Felix
>

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I don't think you are having much luck in Germany. There is one in
Ireland.
If you are interested in starting one we can maybe work together. I
would like to start one Frankfurt (or Hessen if it is to expensive
there). Our Idea was to go for S1 Labs at Schools at first. Maybe we
could work out a concept to start one next year (but this year is
really busy) It would be an advantage to have several Labs and a
community in Germany to deal with legal stuff.

Greets
Sebastian

On 26 Jan., 19:39, Felix Maier <ukai....@googlemail.com> wrote:
> Are there any DIYbio labs in Berlin, Germany to join ?
> Anyone building  up a DIYbio lab for i.e. workshops?
> I am very interested in this subject.
>
> Feel free to contact me via email.
> Felix

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Not very comprehensive, but the I like the title, and it shows that
people do want this type of design/parts library to exist...
http://biomicrofluidics.com/phpbb/

via the UCLA DiCarlo Microfluidics Lab

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Venus is a much more desirable candidate for many reasons:
Mass- Mars isn't big enough to support an atmosphere fit for human
habitation. You could float habitats on Venus' atmosphere.
Productive Energy (thermal and non-ionizing radiation)- Mars' surface
is relatively devoid of non-destructive energy. Venus has too much,
even simple heat pumps would effectively convert atmospheric energy to
other forms.
Destructive Energy (ionizing radiation)- Mars is awash in it. Venus'
atmosphere has a magnetosphere that affords a level of shielding.
Chemistry- Mars has lower water, carbon, and nitrogen in any form on
its surface or in its atmosphere than Earth. Venus has more of all of
the above, typically boiled in acid and at 90 atm of pressure such
that they are unusable to biological systems.
Value- Fixing Venus teaches us lots about "fixing" Earth. Fixing Venus
will be all about collecting energy rather than just expending it.

IMO, put an outpost on the Moon, colonize Mars, terraform Venus.
Anything else is making a purse out of a pig's ear. On that note; the
human body is supremely adapted to this planet and for relatively
short periods of time at that. Bending atmospheres to fit our lungs,
putting chairs in spacecraft to fit our rear ends, and blasting
surgeons and/or medicine around the Solar System is, to me, somewhere
between naive fantasy and religion.

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The latter; new Google Groups Interface included a little screwup that
concealed lots of posts.

On 31/01/12 15:39, Nathan McCorkle wrote:
> This just came to my inbox, but google tells me it was sent 12 days
> ago... am I crazy or did google groups have some error?
>
> On Wed, Jan 18, 2012 at 11:38 PM, Sophia <sandren1@student.gsu.edu> wrote:
>> http://www.facebook.com/DIYbio
>>
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This just came to my inbox, but google tells me it was sent 12 days
ago... am I crazy or did google groups have some error?

On Wed, Jan 18, 2012 at 11:38 PM, Sophia <sandren1@student.gsu.edu> wrote:
> http://www.facebook.com/DIYbio
>
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I think its wrong to say annealing only happens between 50-65 C, but
that's the general range for most primers 15-25 bp in length

On Fri, Jan 27, 2012 at 11:57 AM, veera <drveeramanikandan@gmail.com> wrote:
> generally annealing temperatures will be just 3-4 C below Tm of primers. if
> the melting point of primers is very high like you said in the range of 90 ,
> at that hight tempertaure all double stranded dna will split off and so
> annealing will not take place., because annealing happens only in the range
> of 50 - 65 C. hope i'm correct.. if i'm wrong anyone can correct me...
>
> Jonathan Nesser wrote:
>
>
> Sorry to start yet another new thread for such a small question, but I
> don't want to pollute other threads with off topic posts... I've been
> trying to understand why primer melting temperatures have to be in the
> 55-65C range when PCR cycles call for 94C for DNA melting... I've
> tried to find an explanation for this but haven't been able to...
> Wouldn't that result in the primers splitting off from the template
> DNA before you reach the extension cycle of 75C (or around there)? I'm
> sure I'm wrong because obviously these PCR programs work, but I don't
> know WHY I'm wrong, and would like to understand. Thanks again for
> explaining something that is probably so common knowledge that nobody
> feels the need to address it in writing :)
>
> Jonathan Nesser
> jonathan.nesser@gmail.com <mailto:jonathan.nesser@gmail.com>
> diybioandneurosci.blogspot.com <http://diybioandneurosci.blogspot.com>
>
>
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I'll upload a picture in a while of my current eppie-rack. I drilled it
out of MDF and stuck some bolts through it as legs.

Looks hella ghetto.

On 31/01/12 15:33, Jeswin wrote:
> what do you guys use in your home labs to hold 1.5 ml microcentrifuge
> tubes or 0.2 ml PCR tubes?
>
> I'm going to take the racks the pipet tips come in, flip it over and
> use it as a rack. Works well for small PCR tubes. The stuff you buy
> online looks a bit expenisive for PCR tube racks. THe 1.5 ml racks are
> cheaper though.
>

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Hey Rick,
I think PCR is almost certainly the way to go, for a few reasons.

Firstly, you're talking about mammalian viruses; to grow them (which is
a bad idea in any case), you'd need mammalian cells, and mammalian cells
call for more intensive care than bacteria. It's a big investment, and
it requires a lot of time and effort just to keep them going.

And that's ignoring the fact that each virus will call for a different
target cell type, which in turn calls for different care procedures;
kidney cells may need very different medium and care than liver cells,
for example.

Secondly, plaque assays don't really work, AFAIK, for mammalian viruses.
Because they must be grown with a liquid layer on top, and that liquid
layer allows viruses to spread freely, instead of growing outwards like
bacteriophages.

Besides, if dealing with human infections, a good rule #1 is *never
culture*. If you can do your work without culturing the viruses at all,
then you should.

DNA survives boiling and alcohol, but viruses don't. If you want to
study your kids' colds, you could just take a sample of nasal mucous,
boil it in an eppendorf tube for 10 minutes, add 40% EtOH just to be
sure and then use a tiny sample for PCR, using primers that will amplify
your virus of interest. Ideally use primers that are already tested and
shown to work reliably in the available science literature. If you make
your own, aim for 25 nucleotides in length in a non-repetitive area of
the virus genome that's probably well-conserved, such as promoters.

PCR is great for many reasons. One reason is that it allows you to study
something that is impossible or unwise to culture, such as thermophiles,
bizzarre soil symbiotes, or human pathogens. I would strongly suggest
sticking with PCR and taking normal precautions to avoid infecting
yourself while you take samples.

On 25/01/12 05:51, Rick Byers wrote:
> Hi,
> I'm curious to better understand the pattern of viral infections in my
> family (I've got two young kids, so it's pretty common for some cold
> to be going around our house). I'm trying to figure out the most
> practical way to do regular URT diagnostics. I've got no lab
> experience (but would love to learn) and almost no lab supplies (but
> am willing to invest up to a few thousand dollars as necessary).
> Ideally I'd like something that is both quantitative (so I can track
> viral load over the course of infection) and multiplexed (so I can
> identify which viruses are responsible and catch most common ones).
>
> I know I can do some simple diagnostics at home with a modest
> investment. Eg., I considered doing simple plaque assays or simple
> PCR runs, but I'm hoping I can find something that's less time
> consuming at non-trivial scale (eg. detecting at least 10 different
> viruses/strains, ideally many samples over a period of time). Perhaps
> there's an immunoassay panel of some sort I could use at home? Or
> perhaps the best option is a service that will do a multiplexed qPCR
> run for me on a set of samples I send in.
>
> I'm continuing to do research of various products and diagnostics
> services, but I'm finding it pretty difficult to get specific prices
> and weigh the options. Any advice would be greatly appreciated.
> Thanks!
>

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what do you guys use in your home labs to hold 1.5 ml microcentrifuge
tubes or 0.2 ml PCR tubes?

I'm going to take the racks the pipet tips come in, flip it over and
use it as a rack. Works well for small PCR tubes. The stuff you buy
online looks a bit expenisive for PCR tube racks. THe 1.5 ml racks are
cheaper though.

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I've regularly done something similar for western/immunoblots.  The 'protocol' couldn't be simpler, drain antibody into a falcon and put in freezer.  Next time you want to use it, thaw and place on membrane.

I've no idea if a similarly basic protocol would work for your application though.

Oh, nice idea. Protein recycling; that's one step ahead of the curve!

If the antibody was purified using some peptide-based method, such as
maltose-binding proteins or his-tags, you could try using the same
methods to separate from the finished experiment?

On 18/01/12 16:48, Andrea J Bae wrote:
> Hello Everyone!
> I'm an undergrad at Wellesley College starting immunocytochemistry.
> I'd like to know if there are ways to recycle antibodies for ICC. Does
> anybody have any suggestions from experience or any protocol
> suggestions from the literature?
>
> Thanks!
> Andrea
>

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