Backyard Biosynth

  • 2
  • Article
  • Updated 9 years ago
Ok, following on from the chat.

Components to have your own lab.
Maybe someone out there can find better prices than the ones I can find.
Here's to hoping.

Thermocycler
If someone can find where to buy the Lava Amp, would be most appreciated.
Open PCR - http://openpcr.org/
$512
Coffee Cup Thermocycler - http://www.instructables.com/id/Coffe...
$350
Light Bulb PCR - http://citizensciencequarterly.com/20...
$50
Lava Amp - http://www.newscientist.com/article/d...
$10

Agarose Gel Box
Open Gel Box - http://openwetware.org/wiki/DIYbio:No...
http://www.sciencebuddies.org/science...
Keiki Gels - http://openwetware.org/wiki/DIYbio:No...

Strategene Used Electrophoresis - http://www.medwow.com/used-electropho...
$230
BioCell - http://www.modernbio.com/equipment-su...
$170

Vortex Mixer
I'm thinking if you can find any old blender where you can attach a few tubes will do the trick.
What's the difference between a Vortex Mixer and a Centrifuge?
Vortex - http://www.vortexportablemixer.com/#
Hehe, it'll most probably still do the same job as a proper Vortex Mixer
$20

Magnetic Bead Stand
- Need more info on why this is needed and what it does?
- Limited information online

Buying a lab via Ebay for less than $1000
http://scienceblogs.com/worldsfair/20...

Enjoy!

Maybe it'll be good to know what Das Lab use for Eterna's synthesis?
Photo of Berex NZ

Berex NZ

  • 116 Posts
  • 20 Reply Likes

Posted 9 years ago

  • 2
Photo of chaendryn

chaendryn

  • 29 Posts
  • 1 Reply Like
Awesome, thanks :) Time to go hunting.
Photo of blubblub

blubblub

  • 60 Posts
  • 1 Reply Like
Thanks Berex!

In Wisconsin we have the UW SWAP SHOP (surplus with a purpose) where materials are recycled to the general public after they become obsolesced. Often enough, the old stuff is better made, simpler to operate and understand and just as effective on the fundamentals.

The link is http://www.bussvc.wisc.edu/swap/

One of the categories on the online auction is: Lab/Medical Equipment.

You can bid on and win items for a smidgeon of its actual cost. I would think that most States with a thriving academic community would have something similar. It may be something to check out along with EBAY. Inventory is added on a weekly basis and auctioned off over two to three weeks.

Perhaps some of the items on your list will be on there from time to time, or on other similar 'SWAP' websites.
Photo of blubblub

blubblub

  • 60 Posts
  • 1 Reply Like
Just a quick followup. I went to closed auctions on the UW SWAP Shop link and a DNA Thermal Cycler just sold for $51.00.

Requires standard power (not included)
Part #N801-0100
Features dB-25 serial port
20 character monochrome display
Cosmetic condition: good, minior wear (scratches)
On power up, compressor kicks in
Model #480
Serial #P30496
Manufactured by Perkin-Elmer
120 volts
Unit size: 12.25" H x 13" W x 19" D
No manual
No power cord
Unknown condition
Powers on, otherwise untested

Here are the bids for this auction (highest bids first):
Bidder Bid Amount Date of Bid
jaltwies $51.00 4/18/2011 7:02:01 AM Central Time
kcornwall $50.00 4/17/2011 11:15:33 PM Central Time
Paul Zuelke $45.00 4/12/2011 9:00:22 PM Central Time
Fugent L.L.C. $33.00 4/4/2011 7:17:53 PM Central Time
Photo of chaendryn

chaendryn

  • 29 Posts
  • 1 Reply Like
Contacted the LavaAmp guys to find out whether it's available for sale. Let's see what I hear back from them.
Photo of chaendryn

chaendryn

  • 29 Posts
  • 1 Reply Like
Okays, LavaAmp is not commercially available yet. They hope to have their first units by the end of the year. So we'll have to find another solution until then. But did ask Jim to keep me posted on developments.
Photo of alan.robot

alan.robot

  • 91 Posts
  • 36 Reply Likes
A few comments -

a vortex mixer is just a contraption that you press an eppendorf tube against and it shakes it vigorously. It accomplishes the same thing as when you shake a baby bottle to get the formula to mix with water (heheh, bet you cant guess why that example comes to mind). The link you found is not what was meant - look at this link for an example of what I mean:

http://www.neutecgroup.com/wizard.htm...

It's not a blender or a centrifuge, it's just a vigorous shaker with a momentary on-off switch that activates when you press against it (convenient when you have lots of little tubes to mix). I'm sure you could cook one up yourself, maybe mount a little holder to one of those back massager thingies, add a foot pedal on-off switch or something like that.

The centrifuge is a "big ticket" item for a home lab, since there's really no non-scientific equivalent to jury-rig. You really don't want to home-brew something that can spin 3000 G's on your own, it's just too dangerous. Brand new they cost over a thousand dollars, but you might be able to find used ones for much less, google "benchtop centrifuge".

And ask what it was used it for before, you don't want one from some hospital lab were they were spinning pee or blood samples - stuff always spills . .

You'll have to look at the protocols to make sure you get one that can take the right size test tubes at the right speed for your application. It's often used for separating different substances from each other, for example many kits include purification steps that require you to centrifuge your sample through a provided single-use filter (like immobilized magnetic beads).

Lastly, the "magnetic beads" are from a commercial kit for purifying PCR products: probably something like this:

http://www.beckmangenomics.com/produc...

Most of molecular biology is kit-driven these days, little pre-packaged, pre-measured mystery ingredients in disposable tubes that do things more efficiently than old-school making your own chemical stocks. While that's great for reproducibility (and convenience - you don't need access to a chemical stockroom), kits can be very expensive per reaction so it helps to comparison shop between different kitmakers. I have no idea how expensive the magnetic bead purifying kit is, but I can assure you there are dozens of companies that sell similar products for purifying PCR products if it turns out to be very expensive.

Cheers,
Alan
Photo of rhiju

rhiju, Researcher

  • 403 Posts
  • 123 Reply Likes
I love this thread. I wish it existed when I started my lab. A couple comments to follow up --

1. You don't really need the vortexer. (I never use it!) Its used to make sure that tubes are mixed up when you thaw them after freezing, but you can do that just by shaking them really well.

2. Kits. The magnetic beads are expensive -- one kit costs $400 and works for a few hundred samples. One option would be to buy some beads, and wash them really well with water and 1 M NaCl after each use, and reuse them. My lab is reluctant to do this because we worry about sample cross-contamination, and the companies say you shouldn't do this (of course -- they want you to buy more beads). But I think recycling at least a few times would work...

3. Magnetic stands. The best company for this is VP scientific.

4. The most expensive 'consumable' after kits is reverse transcriptase Superscript III, which is used to read out the chemical modification. A tube of this stuff (which contains little protein machines) can cost several hundred dollars, and can be used for a few hundred reactions. I don't know of a good way to bring this cost down. However there are cheaper reverse transcriptases (AMV reverse transcriptase) that have different properties and probably work well enough for these experiments.

5. For some of the 'equipment'/consumables (96-well plates, tubes, etc.), there are often big 'fire sales' or giveaways when biotech companies go down, which does happen in, for example, the bay area. High school teachers are often on mailing lists that notify them of these event -- might be worth trying to get in the loop.

In reply to a question from Berex and also some other labs around the country, my lab is compiling its explicit protocols and making them public here:

Das Lab RNA synthesis & structure mapping pipeline

These are slightly out of date; we are doing several passes this week, to include updates & tips that we've learned over the last year. So you may want to revisit them in May.
Photo of Ding

Ding

  • 94 Posts
  • 20 Reply Likes
Thanks for sharing all this info!

Just a note: I don't seem to have access to most of the Google Docs linked from your website. I can read "Designing Oligos for DNA templates" and "Confirmation (coarse) of DNA or RNA synthesis with agarose gels" but not the rest.

edit to add: it does give me an option to request permission to read the other documents, but I'm not sure you really want emails from everyone who just wants to take a look...
Photo of alan.robot

alan.robot

  • 91 Posts
  • 36 Reply Likes
Awesome that your lab is publishing protocols online Rhiju!

Just a heads up that the last 4 links on your protocol page are not viewable to outsiders right now.

@Berex: A general comment that almost any enzyme you can buy in a kit you can grow and purify yourself for a few percent of the cost of the pre-packaged variety. If there is one enzyme you need boatloads of, it just takes some elbow grease to make so long as it expresses well in e.coli (most viral proteins do).

First you do need someone to "gift" you some e. coli containing an expression plasmid with the protein of interest on it with a 6x his tag at the end first (most labs I've dealt with are responsive to such requests as a matter of scientific reproducibility so long as it's not patented material). Once you get some, you just grow much as you need.

In fact, this was a half-day exercise in my undergraduate molecular biology lab course (this was back in 2001) to grow and purify some HIV Reverse Transcriptase using just an incubator and a benchtop centifuge.

You mix up some Luria Broth (bacto-tryptone, yeast extract, glucose, and NaCl, all of which you can buy for a few dollars a pound). Innoculate with a toothpick of bacteria, shake and incubate overnight. Add an inducer (IPTG, also cheap), wait a couple more hours, then you just spin down the cells, crack the cells open, and run them over a nickel/cobalt column.

Literally, you drip the broken cell slurry down a tube with the nickel beads in it, only the protein with 6xhis will stick, the rest will fall off after you rinse it

Voila, a test tube full of RT!!

You can purify any protein that has the 6xhis tag at the end by running it over a nickel/cobalt column. You buy a jar of beads coated with nickel/cobalt for not that much (< 100$), and they can be re-used almost indefinitely and are easy to clean.

Here's a comparable protocol from openwetware:

http://openwetware.org/wiki/Knight:Pu...
Photo of rhiju

rhiju, Researcher

  • 403 Posts
  • 123 Reply Likes
I fixed the links -- they are public now.

Also, do look at openwetware.org -- there's a goldmine of information there.
Photo of Berex NZ

Berex NZ

  • 116 Posts
  • 20 Reply Likes
@rhiju is it possible to have a list of each of the consumables used in the process? Including the product codes. I'd like to be able to calculate how much it'd cost and how long they'd last.

Thanks.
Photo of rhiju

rhiju, Researcher

  • 403 Posts
  • 123 Reply Likes
We have the consumables and costs per weekly design cycle compiled somewhere. I will ask Daniel to do a post.
Photo of alan.robot

alan.robot

  • 91 Posts
  • 36 Reply Likes
check out the video protocols on this site:
http://www.benchfly.com/protocols.php
Photo of Berex NZ

Berex NZ

  • 116 Posts
  • 20 Reply Likes
@rhiju just reading those links to the Das lab. First of all, thank you very much for them.

Have a few questions, naturally.
In regards to the Oligos,
How do add the promoter, buffer and tail to the sequence?
Is there any chance of using open source scripts instead of MATLAB?
If that is too time-consuming, if we could be provided with the high def pictures generated from each sample?

What is the spectrophometer (nanodrop) used for? I know its used for measuring light wavelengths, but how does that helps us?

There was a note on a HiTrace manual annotation server, are there still plans for one to go ahead?

This one would more likely be directed to jee, but just wondering what has eterna been built on and whether its possible there are some programmers out there that could help speed up the development process?
Photo of Matt Baumgartner [mpb21]

Matt Baumgartner [mpb21], Alum

  • 128 Posts
  • 33 Reply Likes
Octave is an open source program that can run matlab scrips (for the most part).
http://www.gnu.org/software/octave/
Photo of Berex NZ

Berex NZ

  • 116 Posts
  • 20 Reply Likes
Thank you to alan to picking this up. There is now a stand-alone HiTrace GUI which you don't need MATLAB for anymore.
https://simtk.org/project/xml/downloa...
Photo of alan.robot

alan.robot

  • 91 Posts
  • 36 Reply Likes
Here's a great site that explains what the UV-VIS spectrophotometer does:

http://www.labautopedia.org/mw/index....
Photo of rhiju

rhiju, Researcher

  • 403 Posts
  • 123 Reply Likes
To get a sense of pricing, I'm posting a spreadsheet here that Daniel put together a little while back. It summarizes the total material costs per week for EteRNA, assuming 8 designs/week with lengths of about 100 nucleotides a week. The price is somewhat higher for longer RNAs (e.g., bulged star).

You'll see that the dominant cost is for the DNA for the syntheses. Please also note that we have negotiated lower prices for some of the chemicals/reagents because of our affiliation with Stanford (which, in total, buys a lot of this stuff).

@berex, we didn't include manufacturer/serial numbers/quotes here, but we're trying to include these in the protocols.

Let me know if the link doesn't work; sometimes Google docs sharing settings are problematic.
Photo of Berex NZ

Berex NZ

  • 116 Posts
  • 20 Reply Likes
Hi rhiju,

Thank you for that.
How many oligo's are used per design?
I'm assuming that you're using 25 nmole, which according to IDT, is for sequences between 15-60 nts. Then you have to take into account how long your overlays are going to be. Assume thats about 14nts. Overlays are what is used to join the various oligo's together in the PCR. Then they'd be primers and what not.

For a Branches design that we are on at the moment, its a 119nt design. So I'll assume we need 3 oligos. So break the design up into 45nt blocks. So total it'd be 60, 60, 29 Oligos.

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

IDTDna
25 nmole
Charges 35c per nt in a tube
Charges 18c per nt on a plate

96 well plates, minimum of 24 oligo's
384 well plates, minumum of 96 oligo's

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

Using the above oligos as a measure. 149nts are needed.
Would mean $52.15 per design. Somewhat close to you spreadsheet cost of $40.63 per design.

Will need clarification on the 105bp design though, since that means at least 210 nts are involved. Which makes the maths funny.

Now if you switched to using plates, and we use enough oligo's to warrant it. Instead of $52.15 per design, it would now be $26.82 per design.

Assuming 8 designs, 3 oligos each. Thats already 24 oligo's required. Not to forget the bot designs as well.

Whats interesting, is that Standford also does some freelance oligo synthesizing.
http://cmgm.stanford.edu/pan/rates.ht...
And at plate rates, thats 10c per nt.

It might also be prudent to see if you can get on IDT's Preferred Circle program.
Then you can get rates like John Hopkins (daf.jhmi.edu), where instead of the IDT retail of 18c, you can get it down to 12c. Which would then make it $17.88 per design.

If there are any errors, please let me know.
Photo of rhiju

rhiju, Researcher

  • 403 Posts
  • 123 Reply Likes
Berex, thanks for the breakdown. In actuality, we have a pretty low price with IDT because of how much we buy from them.

Most importantly, we also have a nice courier delivery system that guarantees a less-than-24 hour turnaround between typing the sequences into their website and having it appear on our benches; this is again free because of the large amount of DNA that our lab and Stanford buys from them!

We could go cheaper (to plate rates, $0.09-0.10/base) but we'd incur a delay of about a week from typing in your designs and the DNA getting to us. Your e-mail does make me think whether we should somehow set up an 'eterna'-specific quote -- if you or other garage bioscientists end up purchasing stuff from IDT, let me know!
Photo of Berex NZ

Berex NZ

  • 116 Posts
  • 20 Reply Likes
Thanks for the quick response! :)

Ahh so you'd go for the same Day oligo's. Which retails for 60c per nt, but obviously you are getting it less than that.

Thank you for the clarification. Was a bit scary when I thought I found better pricing than your current pricing structure.

It would be awesome to have an 'eterna' specific quote. You'd just have to be aware that you'd have to get at least 3 quotes. A US one, a AUS one and a NZ one.
But in general, if someone did setup a garage lab in the States, that would be the best and cheapest option at 18c per nt. Would require an amount of their time though. I'm not aware of how long it takes for each design to be processed.

Maybe there could be an additional eterna page. Where people could pay for their designs to be synthesized, and an additonal fee for it to be processed? At the moment, it'd be like $20 per design, but with the falling sequencing costs, could easily fall. When it finally hits the $5 price point, then a lot more synthesis would be able to be subsidised possibly. :)
Photo of Berex NZ

Berex NZ

  • 116 Posts
  • 20 Reply Likes
List of some DNA Sequencing facilities
http://www.nucleics.com/DNA_sequencin...

According to openwetware, this is what is available at Stanford.
I’m assuming this is also what the Das lab are using.
DNA Sequencer ABI 3100, Sequence Detector ABI 7900HT, Spectrophotometer ND-1000
http://openwetware.org/wiki/Stanford_...

Das Lab uses 5' T7 Promoter (20nts), 3' tail Primer (20nts), and a 5' buffer (15nts). Total 55nts are needed to be added to the sequence.

Revision for Branches is 210nts.
@18c is $37.80 USD
@12c is $25.20 USD

@rhiju quick question, what books would you recommend? In general books and some extensive lab prep books.
Photo of merryskies

merryskies

  • 40 Posts
  • 3 Reply Likes
Hello rhiju,

Regarding your comment:"We could go cheaper (to plate rates, $0.09-0.10/base) but we'd incur a delay of about a week from typing in your designs and the DNA getting to us".

Would going to the cheaper plate rate reduce the cost enough to allow for more player's designs to be synthesized per round? And, if so, how many?

I understand that it would mean waiting two weeks (instead of one) for the results.

Regards,
merryskies
Photo of rhiju

rhiju, Researcher

  • 403 Posts
  • 123 Reply Likes
Yes, if we'd wait 2 weeks instead of one we could certainly have 16 made per round, and perhaps 24 ... if there's a consensus among active players that they prefer this route, then we can take it. That cycle could also allow for more time to consider and debate new results before diving into the next cycle!

@Berex, not sure what lab prep books are good -- I learned through hands-on teaching from a great mentor (Rick Russell, now a professor at U.T. Austin) and by checking his notebooks.
Photo of rhiju

rhiju, Researcher

  • 403 Posts
  • 123 Reply Likes
Also, @Berex, the devs have debated opening up the EteRNA pipeline to allow 'pay-for-play'. The main thing we worry about is that EteRNA might start being considered a 'service' rather than what it is: a cutting-edge scientific and social project that is tackling (and, by all the current evidence, *solving*) some fundamental problems in biology and bioengineering.

At the same time, we are working overtime to apply for grants to help us keep EteRNA going. It has sometimes been a tough sell because government agencies are facing tighter budgets and are getting even more conservative about what they will fund.

Obviously if you or others have leads for agencies, private foundations, donors or other funding sources, we'd love to hear about them. We're setting up gift accounts to collect donations for the EteRNA project, but we don't want to ask players to contribute money, since they are already contributing an enormous amount just by playing and posting.
Photo of Berex NZ

Berex NZ

  • 116 Posts
  • 20 Reply Likes
You don't need to ask, some of us are already passionate about the project's premise. Please let me know when the gift accounts become available to donate into.

Thank you.
Photo of chaendryn

chaendryn

  • 29 Posts
  • 1 Reply Like
I second Berex's comment - please let us know where we can donate to help with the project :)
Photo of rhiju

rhiju, Researcher

  • 403 Posts
  • 123 Reply Likes
Thanks again for the support ... we just set up an EteRNA Research Fund that allows donations to the project that we will use for syntheses. We put a little blurb in 'about eterna'. The link to the actual 'giving' website is on there as well [Direct link here.]

Again, I want to emphasize that is not our expectation that players donate money to keep EteRNA going --you are already contributing greatly by playing and posting!
Photo of merryskies

merryskies

  • 40 Posts
  • 3 Reply Likes
Hi rhiju,

Regarding your response: "Yes, if we'd wait 2 weeks instead of one we could certainly have 16 made per round, and perhaps 24 ... if there's a consensus among active players that they prefer this route, then we can take it. That cycle could also allow for more time to consider and debate new results before diving into the next cycle!"

It’s ok with me if my designs are not synthesized. I can still play and learn from the synthesis results. But the option of having a larger number of player’s designs synthesized per round is very appealing for several reasons.

I am aware that major changes to the voting system are planned to be implemented soon. Perhaps sometime after the new system has been introduced and tested, then this topic could be put forth to the players for further discussion.

Thanks for your response,
merryskies
Photo of alan.robot

alan.robot

  • 91 Posts
  • 36 Reply Likes
I second Merry's point, I do feel strongly that the limitation of 8 slots/week is pretty much constraining the number of super-active designers to around a dozen or so, and that's not something that even a perfect voting system would "fix". It usually takes several iterative designs for people to test out their ideas fully enough that they gain some intuition rather than an isolated one shot in the dark, something that is just difficult with so few slots.

Perhaps the two-week turnaround could be compensated by staggering two, independent design challenges separated by one week? Just a thought. I think that the self-sustaining "critical mass" in terms of the number of super-active lab participants has not been reached yet. More discussion of designs and feedback certainly would not be a bad thing - if too many people are "throwing together" a design at the last minute to meet an arbitrary deadline it dilutes the cool stuff learned. . .
Photo of Berex NZ

Berex NZ

  • 116 Posts
  • 20 Reply Likes
Hi rhiju,

Hmm, well if we could do 24 every 2 weeks, might be better overall. Although a mix of same day and normal plates might be better. That way you have some samples to work on while waiting for the rest, although of course that will most probably require a bit more administration. But that would allow the results to trickle out as they get processed, so the in-between period will seem less obvious.
You could get the players to vote... or the eterna team could just make an executive decision as to what way they want to go.

Hehe we all arent that lucky to have access to a practical mentor and his notebooks. But if you ever come across any good books, please let me know. ;)

I was thinking along the lines of 'pay-for-service.' Like you'd keep everything as it is right now, but for those willing to pay, then their designs would also join the synthesis queue as additional secondary priority. Main priority is to process the main eterna designs.
Thats why I added the caveat, who knows who will take it up. It would be somewhat simpler if a player started up their own independent lab for the service, but there isn't enough documentation to see how viable that is yet.
Who knows the 'pay-for-service' might even be able to subsidise the main eterna synthesis program. Now that would be cool :)
Photo of alan.robot

alan.robot

  • 91 Posts
  • 36 Reply Likes
http://cshlpress.com/default.tpl?cart...

I haven't actually read this book but the TOC and reviews look good. Nothing can substitute for having access to an actual molecular biologist to show you the ropes, of course. But even then, most will not be super-familiar with RNA specific concerns (like the need for special RNAse free chemicals, water, lab supplies etc), hence the need for a book like this.

Cheers,
Alan