Re: [DIYbio] Hacking Stink Bugs

[Jeswin]

Nice setup but do you have a UV lightbox to view the stained
electrophoresis gel?

Anyway, you can start with the primer design using BLAST to find the
appropriate sites.

As to isolation, is there anything different in insects that could
mess with purification of DNA?

And talking with suppliers, maybe you can say you are home-schooling
and working on this project. Maybe they can help you out if its some
kind of educational related.

On Thu, Dec 27, 2012 at 12:49 PM, Sticky Ends <juleso...@gmail.com> wrote:
> Greetings,
>
>
>
> My 12 year-old daughter wants to be a molecular biologist. Sounds like
> quality time to me. I'm all in.
>
>
>
> The project is RFLP of stink bug genomic DNA (Halyomorpha halys). We not
> looking towards cutting edge research. We'll be stoked if we can isolate,
> restrict, electrophorese and visualize.
>
>
>
> I've collected some nice equipment including an electrophoresis system,
> centrifuge, pipettor, UV spectrophotometer and precision balance. All that
> wasn't cheap but I really want this project to work because success will be
> inspirational to the lass.
>
>
>
> Some questions to the community if I may?
>
>
>
> I plan to use spin-column kits for DNA isolation. In particular I like the
> EZNA kit from Omega Bio-tek. Any comment on this choice?
>
>
>
> Reagents are proving difficult to acquire. I understand why suppliers
> frustrate the process. But surely there is an avenue to quality reagents
> that is controlled yet accessible. Any advice on how to inform suppliers?
>
>
>
> Finally, as we are beginners, any other advice on getting started will be
> appreciated.
>
>
>
> Best regards,
>
>
>
> The Sticky Ends
>
> Maryland, USA
>
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>

[Dakota]

Pretty cool stuff!  

I was going to recommend Epoch for their spin kits, I have 3 of them and they all work great, plus they seem to be 50%+ cheaper than other places I've found comparable spin kits.

http://www.epochlifescience.com/Product/Default.aspx

Though I did look up the Omega Bio-Tek genomic isolation spin kit, and it's nice they sell them in packs of 5, so that'd probably be enough and save ya some cash.

Other than that for a gel all you really need is agarose to run a gel + a DNA stain.  I recommend GelGreen which can be bought for $21 for enough to make 50 50mL mini gels

http://www.phenixresearch.com/products/rgb-4105t-gelgreen-nucleic-acid-stain.asp

I use it and it looks great under UV light.  http://gyazo.com/6282a5952c347fb453a1dbefa94276b5

Make sure you have UV filtering glasses or a cover before viewing it or you'll hurt your eyes.

It also works with blue LED's but...havn't tried that yet because I had the wrong colors hanging around.

Can also grab agarose and any buffers you need from phenix.  I used them for gelgreen + agarose.  agarose is $50 i think for 25g, again, enough for ~50 mini gels.  Throw in some tips and 1.5mL centriuge tubes if you need them.  You're also going to need some non-denatured high % ethanol, which will probably be the single hardest item to get.  The spin kits require 96%-100% ethanol non-denatured ethanol in 1 or 2 of the buffers generally.  Would using denatured ethanol hurt the DNA recovery?  I have no idea, but I know it's not recommended, so maybe someone else can comment.

http://www.phenixresearch.com/products/electrophoresis.asp

Other than that all I can think of you needing are restriction enzymes...try www.neb.com  You could say are a home-schooler and maybe they'll wave the restrictions they sometimes place on residential addresses.  Otherwise , maybe see if the science teacher at your daughter's school (or local high school) would be willing to order the science stuff to go there, and if you and your daughter get it to work, she can show it to the class and perhaps make a classroom experiment out of it.  

There are lots of supplies out there but after a little shoppingp phenix seemed to have most/all of what was needed at a decent price & free shipping.  If you find a better place let us know!

So based on what you said you'll probably still need.

-pipette tips

-1.5mL microcentrifuge tubes

-agarose

-GelGreen

-DNA loading dye

-DNA ladder

-95%+ non-denatured ethanol (no idea)

-UV protective cover for gel box or UV protective glasses

-Restriction enzymes (new england biolabs, or ask local highschool, I know AP bio students generally always do a restriction analysis for one of the labs).

[Sticky Ends]

phillyj and Dakota,

 

Thanks for your timely and informative replies.

 

The EZNA kit employs proteinase K which, as I understand it, acts in both lysis and protection from DNA degradation.  Will you comment on that phillyj?

 

Another consideration to the EZNA kit is chloroform.  That reagent is added to "remove much of the polysaccharides and proteins and improve spin-column performance."  Regarding chloroform what I know is:

 

PVA gloves should be used in handling

As a gas it's exposure limit is 50ppm per 8 hour day.

It may decompose to phosgene in the presence of oxygen.

Stabilized by alcohol it has a shelf life of no more that 1 year.

Disposal is regulated

 

Anyone care to share their experience with chloroform?

 

My plan is to aliquot 500uL containers as that is the volume prescribed by the EZNA protocol.  I'll do that under a fume hood in the lab.  Having minimal aliquots should reduce the health and environmental risk given our amateur environment.  Comments?

[Sticky Ends]

Arnold,

 

This is our original project idea at least regarding the brown marmorated stink bug, a recently invasive species from China.  Otherwise I think it's reasonably conventional stuff as might be found in a high school AP biology class.

 

We'll be sure to post all of our details here and, hopefully, make a path for you to follow and improve.

[Sebastian S. Cocioba]

Buying the ingredients for spin column reagents from scratch is cheap and easy. Ebay is amazing for this. If you go to the openwetware page there are some recipes for the solutions. I bought my columns from sydlabs and once can regenerate the columns with HCL (ebay for 37% @ 500mL). I get good yields of around 200ug per miniprep (verified with Qubit fluorometer) when substituting guanidium hcl with urea at same molar concentrations. Chloroform is just to regulated and dangerous...but the results are almost unbeatable. Good luck! If anyone is interested I can post my recipes for P1, P2, etc etc. its very similar to Sigma's stock recipe.

Sebastian S Cocioba

CEO & Founder

New York Botanics, LLC

Sent via Mobile E-Mail 

On Dec 27, 2012, at 12:49 PM, Sticky Ends <juleso...@gmail.com> wrote:

Greetings,

 

My 12 year-old daughter wants to be a molecular biologist. Sounds like quality time to me. I'm all in.

 

The project is RFLP of stink bug genomic DNA (Halyomorpha halys). We not looking towards cutting edge research. We'll be stoked if we can isolate, restrict, electrophorese and visualize.

 

I've collected some nice equipment including an electrophoresis system, centrifuge, pipettor, UV spectrophotometer and precision balance.  All that wasn't cheap but I really want this project to work because success will be inspirational to the lass.

 

Some questions to the community if I may?

 

I plan to use spin-column kits for DNA isolation.  In particular I like the EZNA kit from Omega Bio-tek.  Any comment on this choice?

 

Reagents are proving difficult to acquire.  I understand why suppliers frustrate the process.  But surely there is an avenue to quality reagents that is controlled yet accessible.  Any advice on how to inform suppliers?

 

Finally, as we are beginners, any other advice on getting started will be appreciated.

 

Best regards,

 

The Sticky Ends

Maryland, USA

--

[Sticky Ends]

Sebastian,

 

Thanks for the referral to OpenWetWare.  Lots of information there.

 

We're going to try some manufactured kits first.  Then we'll definitely want to discuss hacker spin columns.

 

I read you when it comes to chloroform.  My guess is the Omega kit will suit our purposes even without applications of chloroform.  So maybe we'll go that route.  Anyone care to comment?

[Dakota]

http://www.promega.com/products/dna-and-rna-purification/genomic-dna-purification-kits/wizard-genomic-dna-purification-kit/

is another option, it uses a centrifuge @ 13k but doesn't need silica resin columns, and I think you don't need to provide any extra solvent to the buffers, but not positive.  In all honestly the best thing you can do is just give it a try.  I've found myself dragging on projects for to long, months sometimes, pondering if this will work or that will work, and in reality, all those questions could be answered in 1 day in the lab vs 30 pouring over protocols, books, and forums.  Obviously, making an informed decision from the get go drastically increases your chances of getting things done (with good results), but when in doubt, spend time in the lab.  At 16$ for 5 gDNA isolations, that Omega kit seems fine, and if not, oh well you're only out the cost of a large pizza.

[Sticky Ends]

Everyone,

 

May I thank you once again for your replies.  Great to have your advice and to find a community of like-minded folk.

 

May I raise another topic?

 

Preparations of insect tissue are ideally made by cryogenically freezing and then grinding samples.  I can achieve that but prefer the lesser alternatives.  Any success stories using other mechanical methods such as straight mortar / pestle, coffee grinders or Dounce homogenizers?

 

Note my samples are insects with sturdy exoskeletons.

[Dakota]

If you look at the gyazo link I posted in my first response, that PCR was a result of genomic DNA taken from a mushroom.  The initial sample size was maybe 1/2 the size of a pea, and I ground it for ~30 seconds with the end of a spatula in a 1.5 mL centriuge tube.  There was still tons of debri left over during parts of the extraction (pipette as little debris as possible), but it was taken out after the first spin down. Ended up with a lot of decent genomic DNA from a tiny sample.  Liquid nitrogen was going to be used, but the NMR tank was empty so...didn't get to use it.

I don't think liquid N2 is necessary, or for that matter even dry ice and acetone.  

I see those dead pine beetle/stink bugs all the time at my dad's house up in maine.  They do smell...strange!  It smells like a mixture of ammonia and orange peels / citrus.  Very cool, but it'd be awesome to GCMS it and see what it's made of!

For insects they say even a ground up leg has enough DNA..  Though I just remembered you aren't doing PCR....so perhaps cryogenic freezing prior to the extraction will increase yields. It may still be avoided though, give gDNA extraction without freezing the cells prior and see how much you get.  I imagine it will be enough to do the experiments you want to.  Out of a 200uL final elution volume of gDNA, only 2uL was used in the PCR rxn, so there's a lot left over.  I don't know what kind of amounts of DNA a RFLP uses though, had to look up that acronym.

[Nathan McCorkle]

Proteinase K is a protease, an enzyme that breaks apart amino acid chains similar to a restriction enzyme breaking DNA. Not only will it break down proteins that will generally just clog up the reaction, it wil break down nuclease enzyme that can quickly degrade your DNA. Basic solutions will also aid protein denaturation, and DNA will survive (but not RNA). Phenol/Chloroform washes are awesome for getting clean nucleic acids, but they're a little volatile and toxic, but I don't think much more than common automotive solvents and chemicals. Just wear gloves and long sleeve lab coat, and do the extraction in a fume hood. You probably don't DNA this clean, but it won't hurt if you choose to go that route. The phenol really helps to denature proteins and the chloroform is non-ionic so it doesn't mix much with water despite being polar. Adding some isoamyl alcohol prevents phosgene evolution, wiki mentions using ethanol or amylene, but ethanol is much more water-soluble than isoamyl alcohol.

http://www.worthington-biochem.com/PROK/default.html

There was some talk regarding a paper that showed good results substituting DCM for chloroform...

http://www.scribd.com/doc/51084375/Using-DCM-Instead-of-Chloroform

This may also be of help for the spin-column reagents

http://wiki.biohackers.la/Miniprep

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--
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[Jeswin]

On Thu, Dec 27, 2012 at 9:46 PM, Sticky Ends <juleso...@gmail.com> wrote:
>
> Preparations of insect tissue are ideally made by cryogenically freezing and
> then grinding samples. I can achieve that but prefer the lesser
> alternatives. Any success stories using other mechanical methods such as
> straight mortar / pestle, coffee grinders or Dounce homogenizers?
>

I was thinking about this. N2 is usually a very fast way to grind them
down to a powder that you can extract DNA from. I think getting the
dead dry bugs like Dakota said will be just as good. No N2 needed,
just grind them down. I extracted DNA from 20mg of N2 crushed flax
plants. I did find that getting the right primers is difficult since
many regions are conserved. Finding that region that is unique to that
organism may or may not be trivial.

[shamrock]

Sticky Ends,

 

I noticed that you are located in Maryland, USA. If you're near Baltimore you might want to check out the BUGSS lab (www.bugssonline.org). They can probably help you out with equipment, reagents, and advice.

 

Good Luck-sounds like a cool experiment.  

On Thursday, December 27, 2012 12:49:11 PM UTC-5, Sticky Ends wrote:

Greetings,

 

My 12 year-old daughter wants to be a molecular biologist. Sounds like quality time to me. I'm all in.

 

The project is RFLP of stink bug genomic DNA (Halyomorpha halys). We not looking towards cutting edge research. We'll be stoked if we can isolate, restrict, electrophorese and visualize.

 

I've collected some nice equipment including an electrophoresis system, centrifuge, pipettor, UV spectrophotometer and precision balance.  All that wasn't cheap but I really want this project to work because success will be inspirational to the lass.

 

Some questions to the community if I may?

 

I plan to use spin-column kits for DNA isolation.  In particular I like the EZNA kit from Omega Bio-tek.  Any comment on this choice?

 

Reagents are proving difficult to acquire.  I understand why suppliers frustrate the process.  But surely there is an avenue to quality reagents that is controlled yet accessible.  Any advice on how to inform suppliers?

 

Finally, as we are beginners, any other advice on getting started will be appreciated.

 

Best regards,

 

The Sticky Ends

Maryland, USA

[Sticky Ends]

Gents,

 

Thanks again for the great leads and advice.  BUGSS looks like an awesome space.  Too bad you have to be 18 to participate.  My daughter is just 12.  And this one is for her.  Still, we'll contact them as in community.

 

As to chloroform I have some numbers for consideration.  What volume of chloroform exceeds exposure limits when left to completely evaporate in a sealed 8x8x10 room?

 

(A)  The absolute max permissible exposure limit according to OSHA is 240mg /m3.  So that would require 3.3mL.

 

(B)  The recommended threshold limit value according to ACGIH is 50mg /m3 in an 8 hour workday and 40 hours per week..  Here 0.78mL are required.

 

(C)  Finally NIOSH has established 10mg /m3 as a recommended exposure limit over 1 hour.  Just 0.148mL are required.

 

Good stuff yes.  But it's use clearly demands caution, a chemical fume hood and proper disposal.  So we'll be reading Mr. McCorkle's paper with interest.

[Dakota]

I would avoid phenol/chloroform/isoamyl for at home use.  There isn't a need for it if you have spin kits or other DNA isolation kits.  Spin kits can give you clean DNA without exposure to nasty organic solvents, and take no time at all to do.  I just poured over the MSDS sheets for the Omega Bio-tek kit as well as the Wizard Genomic from Promega which I mentioned earlier.

The only dangerous components listed on the Wizard kit were Tris, which is an irritant, and Ammonium Acetate, which is also an irritant.   It's an isolation kit that doesn't use silica columns.

http://www.promega.com/products/dna-and-rna-purification/genomic-dna-purification-kits/wizard-genomic-dna-purification-kit/

As for extra needed solvents and equipment, this was listed in the protocol handbook.

• sterile 1.5ml microcentrifuge tubes (for 300µl blood samples)

• sterile 15ml centrifuge tubes (for 3ml blood samples)

• water bath, 37°C

• isopropanol, room temperature

• 70% ethanol, room temperature

• water bath, 65°C (optional, for rapid DNA rehydration)

the 70% ethanol may or may not be easier/cheaper to get than the 100% ethanol required for the Omega spin kit.  

The Omega spin kit you listed, if you look at their MSDS, http://www.omegabiotek.com/files/resource/Msds/69614600.pdf

then hit CTRL F to do a search function and type in "hazardous component" you will see there are 6 hits.

Omega seems to use some other things, guanidine hydrochloride, NaOH, sodium dodecyl sulfate (SDS), and then there is this listed in their Elution buffer MSDS

You also need 100% ethanol (95%+ works) to add to their spin kit solutions, but again, non denatured.

Hazardous Components: According to OSHR 29 CFR & 1910.1200, any mixture that contains less 

than 1% by weight or volume of a non-carcinogenic hazardous 

component is not considered hazardous, unless there is evidence to the

contrary.  We do not consider Solution I to be hazardous.  However 

gloves, lab coats and protective eyewear are recommended when using any chemical reagents.

They also have this in their wash buffer - PNP, I think might be this http://www.chemicalland21.com/industrialchem/solalc/PROPYLENE%20GLYCOL%20MONOPROPYL%20ETHER.htm

According to OSHR 29 CFR & 1910.1200, any mixture that contains less 

than 1% by weight or volume of a non-carcinogenic hazardous 

component is not considered hazardous, unless there is evidence to the

contrary.  We do not consider PNP to be hazardous.  However 

gloves, lab coats and protective eyewear are recommended when using

any chemical reagents.

========

To be fair, MSDS's usually make things out to sound 100x more nasty then they really are.  I think even water's MSDS could say deadly if enough is ingested.  They are good to look at though, as they let you know what is in the reagents you plan on purchasing.   It is better to be over-informed then under informed.  

This is one of the legitimate concerns some people have about at home science, maybe not referring entirely to DNA spin kits but....how are people going to handle waste disposal and exposure?  Granted, I've seen people dumping out gas in their back yard and leaving buckets of transmission fluid in a corner for years letting rainwater slowly wash it around, so dumping 500uL of a DNA isolation solution down the drain pales in comparison, but is it still wrong to do?

Furthermore, I doubt a legit chemical waste disposal company would even take waste from an individual.  I was under the impression you needed to be a school or business and setup a contract, but I don't know.  Maybe your DPW's annual hazardous waste cleanup day?

I'd get in touch with a local high school teacher, maybe they would let you and your daughter use the labspace at the highschool some weekend, you can bring in your equipment, and use their waste stream.  You'd get to teach your daughter things in a safe lab place, and the teacher might get a brand new experiment to teach his/her bio class. 

You knew what equipment to buy off the bat and your name is sticky ends, so I assume you know a bit about molecular bio?

  

[Sticky Ends]

Dakota,

 

You put a lot into that last reply and we wish to acknowledge that with gratitude.

 

You sold us.  We recognize the Wizard kit as a better alternative.  We'll give that a whirl if you'll pardon the pun.

 

We also appreciate your comments regarding safety and responsibility.  These sort of opportunities will be closed the first time one of us acts irresponsibly.  We'll do our part.

 

You bring a point I wish to explore, hopefully, without derailing this string.  How should we dispose of our waste?  May I share our plan for review?

 

1.  Recordkeeping of reagent use and generated waste by weight.

2.  All streams of waste containerized and labeled.

3.  Containers bagged by group.

4.  Bags stored in labeled steel drums filled by vermiculite and closed with snap-ring lids.

5.  Quarterly disposal at a licensed facility.

 

Under that regimen we believe we qualify as "conditionally exempt small quantity generators of hazardous waste."  Anyone care to comment on that?

[Dakota]

You are welcome, no problem, glad to help.  I hope it didn't come off like I was trying to use MSDS fear propaganda to push you away from the spin columns.  The concentrations and amounts used in both kits is by no means deadly, and the worst either of them might give you if a large amount was spilled on your skin is probably an annoying rash until washed with soap and water thoroughly.  

For all I know, the Omega kit is more safe than the Wizard Promega kit.   At first I was worried the wizard kit might not give enough DNA, but after checking their protocol handbook.

http://gyazo.com/344d0c323031ca6894614eb0979ecd62

You can see that insect cells give 16ug.  Compare that to a silica spin column which generally binds ~20ug DNA, and they are pretty similar.

I've only used spin kits before, and I'm sure what Wizard has made is a DNA extraction kit that mimics chloroform/isoamyl but without using those things, and without using silica columns.  Spin columns might provide cleaner DNA to be honest, but I've never used the Wizard kit, so I can't compare.

As I see it, disregarding the chemical makeup of the solutions since handling and disposal will be treated equally

Omega Spin Kit 

Pros:

5 isolation reactions for $16

silica binding column, greater DNA recovery and possibly "cleaner" than Wizard

Cons:

Require 100% non denatured ethanol to bring solutions to volume

Wizard non-spin kit

Pros: 

Doesn't use silica columns (if you can call that a pro?  I guess it'd make it cheaper)

Cons:

Needs 70% non-denatured ethanol 

Needs 100% isopropanol

----------------------

So putting the MSDS stuff aside, because I don't want that to be the main factor, I honestly would go with the 5 reactions from Omega to start.  It's $16 vs $150.  I doubt you'll be needing a full Wizard kit with 100 isolations, so then having all that stuff lying around might even offset any danger one might feel reading the Omega spin kits MSDS.

You could use up the 5 spin kits from Omega and get your results, and not have leftover reagents.

Also, I just found a place where it seems you can get non-denatured ethanol for relativley cheap.

http://www.artchemicals.com/Ethanol_Alcohol_190_Proof_NON_Denatured_p/807301608.htm?1=1&CartID=0

I think 25mL or 50 mL would be fine, because the Omega kit I think calls for 6 mL per 2 solvents or something like that, check out the protocol handbook on their website.

You can also get 190 proof alcohol at a liquor store called Everclear, though I don't know if it has other things in it that would screw up DNA recovery.

Spin kits generally call for 96%-100% ethanol to be used (non-denatured is key) and I can't imagine 95% would destroy your DNA recovery.  

Also!!! I just remembered, the Wizard kit has you precipitate the final DNA in 100% isopropanol, then dry out the DNA to dryness, then re-suspend in a DNA re-hydration solution. 

 That solution has EDTA which could chelate to Mg2++ cofactor ions during your restriction enzyme digest and mess with results.

The pro about the spin kit is that you can elute into just plane water, which won't mess up your restriction digest.  

======================================================

So to re-iterate, the 5 isolations for $16 + $30 for 190 proof ethanol would be a better buy then a $150 kit which probably won't ever be fully used, + even more money for 100% isopropanol.

[Dakota]

And to speak to your question on waste, you raised a question I've been wondering for a long time, and also taught me what a "conditionally exempt small producer" was.  I had never heard of the term before.

What you said about properly labeling and separating waste is a good idea.  The only real waste I could see you guys making would be directly related to the solutions involved in the DNA isolation kits, as well as buffer after running a gel, which honestly isn't that bad. 

I just found this link, https://bluetigerportal.lincolnu.edu/c/document_library/get_file?uuid=d4600066-aa07-421e-81f1-95cc163af191&groupId=375800

which shows how many things can be thrown out in the trash or flushed down the drain.

Agarose is on there, Tris is on there, ammonium acetate if neutralized, or brought within range of 5.5-10.5 pH + tons more.

Also, GelGreen can be thrown out in the trash to, http://www.biotium.com/product/product_info/newproduct/gelred_gelgreen.asp

It's different for each city and state, because some cities use septic, some use some other water treatment facility.  Call your local waste management and ask or maybe your police department has one of those links above.  On one hand, one could speculate DIY people don't want to draw attention to themselves by asking about "disposing of scientific waste", but on the other hand, the waste they are generating could be really cheap to dispose of at a facility, if not completely legal to just wash down the drain or throw away.  I don't have a good answer for that because it's different in each state.

Your write-up on proper storage seems fine, if not overkill.  Millions of people have solvents and gasoline and all sorts of stuff sitting under sinks or in sheds or garages not "properly" stored, so I can't imagine getting flak for some salt buffer and ethanol.  

[Nathan McCorkle]

16oz 100% ethanol is $4.79 with a 'tax exempt' permit, how does one get that? Does anyone here have that?

the same bottle shows $17.06 with tax (and you have to buy a case of 24 in either case)

https://us.vwr.com/store/catalog/product.jsp?product_id=7756054

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[Nathan McCorkle]

actually it looks like drying it with zeolite is really cheap

"

Ken wrote ...

... I finally have something to report, namely, how BEAUTIFULLY zeolite (aka "molecular sieve") works to suck the last bit of water out of distilled ethanol. I got a sample of Type 3A Molecular Sieve from a place in So. Calif. called Adcoa: http://www.thomasregister.com/olc/adcoa/molecula.htm

I got a can of the 4-8 mesh -- little balls of rock about 1/8" dia. -- they absorb about 20% of their weight of water over the course of a few hours. Take a liter of 95% ethanol, throw in 250g of stuff, swirl occasionally, filter out the next day thru a strainer, and presto! Anhydrous ethanol. Not expensive either -- $2.05 a pound US in 10 lb quantities, and reusable indefinitely. You drive off the water under a broiler for an hour.

"

from:

http://homedistiller.org/distill/polish/dry

--
-Nathan

[Nathan McCorkle]

sorry, didn't realize that link didnt work anymore, here is the direct link to the manufacturer:

http://catalog.adcoa.net/category/molecular-sieves?

--
-Nathan

[Dakota]

I was always under the impression that denatured ethanol = cheap as heck, but keep the same % ethanol and keep it non-denatured, tax goes through the roof because it could be consumed.  I'm sure people would be buying moonshine straight from VWR if they could get it.  In fact I heard of a professor who used to drink it all the time...as it seems scientists are probably one of its biggest consumers, for lab-work of course.   And I'm also guessing being a state university might make you tax exempt possibly, since it will be used for educational purposes only.  

But, I didn't know you could buy Everclear from a liquor store at 190 proof...that's molecular biology grade stuff!  

Zeolite seems like a clever idea.

[Sticky Ends]

Everyone,

 

We were able to obtain chemicals including pure ethanol without much hassle.   And yes, the ethanol cost a whopping $100 / liter.  Our experience so far is suppliers require two basic responsibilities from their customers:

 

1.  Statement of intended use.

2.  Commercial shipping address.

 

All that makes perfect sense to us.

 

Suppliers have an obligation to protect their enterprise from liability by recording customer statements on use.  Every supplier has called us directly to asses our intentions or knowledge of the products.

 

When they call you must answer their questions truthfully.  Suppose you buy ethanol under the pretense of molecular biology and then divert that material to beverages.  You have now committed mail / wire fraud, a serious crime.

 

Providing a commercial shipping address is a common-sense safety precaution that assures chain of custody in shipping hazardous goods.  Suppose, as you are at work, the neighborhood delinquents snatch that box of reagents from the front porch.

[Nathan McCorkle]

If you have the required commercial shipping address, you should have only paid ~$35 for a liter of tax-included ethanol....

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[Sticky Ends]

Nathan,

 

Having access to a commercial shipping address does not allow one to purchase tax free.  To lawfully purchase tax free you must first make an application to the ALCOHOL AND TOBACCO TAX AND TRADE BUREAU (TTB).  The application form is here:

 

http://www.ttb.gov/forms/f515022.pdf

 

Understand, however, that you will be granting TTB a right of entry to inspect goods and records.  We don't know about you but we would rather pay the tax, about $15 per proof-gallon.  A reasonable compromise between responsibility and privacy.

[Sticky Ends]

And who, may I ask, sells liters of 200-proof for $35 tax paid?

[Nathan McCorkle]

I specifically mentioned tax-free and tax-included versions, along with a link to pricing for both

On Fri, Dec 28, 2012 at 8:05 PM, Nathan McCorkle <nmz...@gmail.com> wrote:

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-Nathan