Backyard Biosynth

General Science Discussion
1

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?

2

Awesome, thanks :slight_smile: Time to go hunting.

3

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.

4

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

5

Contacted the LavaAmp guys to find out whether it’s available for sale. Let’s see what I hear back from them.

6

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.

7

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

8

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.

10

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…

11

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…

12

I fixed the links – they are public now.

Also, do look at openwetware.org – there’s a goldmine of information there.

13

@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.

14

We have the consumables and costs per weekly design cycle compiled somewhere. I will ask Daniel to do a post.

15

check out the video protocols on this site:
http://www.benchfly.com/protocols.php

16

@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?

17

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…

18

Here’s a great site that explains what the UV-VIS spectrophotometer does:

http://www.labautopedia.org/mw/index…

20

Octave is an open source program that can run matlab scrips (for the most part).
http://www.gnu.org/software/octave/

21

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.

22

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.