Quick rough notes

August 27, 2013
1)  Strawberries probably have xanthosine biosynthesis pathway, but I need to double check that they do.
2)  Need to verify the NCBI ascension numbers i have for the caffeine biosynthesis pathway are correct.
After 1+2 then I need to make some primers.
NOTES:
 Xanthosine biosynthesis:
guanine
[e] guanine deaminase
xanthosine
or
hypoxanthine
[e]xanthine oxidoreductase
xanthosine
Caffeine synthase pathway:
XANTHOSINE
[e1] xanthosine methyltransferase (XMT)
7-METHYLXANTHOSINE
[e2] 7-methylxanthosine nucleosidase
7-METHYLXANTHINE
[e3] 7-methylxanthine methyltransferase (MXMT or theobromine synthase)
3,7-dimethylxanthine (theobromine)
[e4] 3,7-dimethylxanthine methyltransferase (caffeine synthase)
1,3,7-trimethylxanthine (caffeine)
Xanthosine biosynthesis:
guanine
[e] guanine deaminase
xanthosine
or
hypoxanthine
[e]xanthine oxidoreductase
xanthosine
Caffeine synthase pathway:
XANTHOSINE
[e1] xanthosine methyltransferase (XMT)
7-METHYLXANTHOSINE
[e2] 7-methylxanthosine nucleosidase
7-METHYLXANTHINE
[e3] 7-methylxanthine methyltransferase (MXMT or theobromine synthase)
3,7-dimethylxanthine (theobromine)
[e4] 3,7-dimethylxanthine methyltransferase (caffeine synthase)
1,3,7-trimethylxanthine (caffeine)
Source:
Uefuji H, Ogita S, Yamaguchi Y, Koizumi N, Sano H. Molecular cloning and functional characterization of three distinct N-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. Plant Physiol. 2003;132:372–380.
Uefuji H, Ogita S, Yamaguchi Y, Koizumi N, Sano H. Molecular cloning and functional characterization of three distinct N-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. Plant Physiol. 2003;132:372–380.

Hangout is live

February 2, 2013

Hangout is live

https://plus.google.com/111857545829837898782

Google+ hangout

February 2, 2013

There will be a Google Plus hangout tonight about anything DIYbio or bio related.  A link will follow.

Science Hack Day 2012

October 16, 2012

I am going to the annual Science Hack Day in San Francisco again this year (November 3-4).  For those of you not familiar with it, its a 2 day event (overnight if you like) where a bunch of like minded citizen science folks get together and work on projects.  Some hacks relate to space and others to biology.

http://sf.sciencehackday.com/

 I’ll be working on two biology projects ( I think).  I am going to try to make a sterile hood out of home depot and target supplies and I am going to work on some PCR related stuff.

 

Dont live near SF?  There are other affiliated science hack days that occur world-wide!  So check it out.

Altoids tins are autoclavable

July 11, 2012

Last night I decided to autoclave (sterilization process that uses 125 C steam) the tin container that Altoids mints come in in order to see if they would survive the temperature/pressure.   The tins looked the exact same inside and outside after autoclaving.

It is not a huge surprize the tins survived – they are metal after all.  My actual intent in doing this was to use the tins as petri-dishes – which is why I needed to sterilize them in an autoclave.  But while the autoclaving of the tins went flawlessly, my media preparation failed miserably.  I tried to make some growth media with red wine, body-builder amino-acid supplements, sugar, and agar-agar (edit: FYI I boiled off most of the alcohol).  For whatever reason the media would not harden.  I will try again sometime after this week because I will be going to the TAM conference in Vegas this year!

Altoids (before)

Altoids (before)

Altoids inside (before)

Altoids inside (before)

As far as using these containers as petri-dishes goes – my only concern is the hinge.  It appears that when closed, the lid covers the hinge hole and should keep the innards sterile.  Until I get the media to harden I will not know if that is a problem or not.

Even if the tins fail to work as petri-dishes (metal could have unforeseen issues for micro-organisms), they may work out as containers for auto-claving parts or components.

Altoids (after)

Altoids (after)

A quick and dirty microbiology attempt

July 3, 2012

I recently felt compelled to do some microbiology work.  My goal was to make a crude growth media for microorganisms, swab my mouth for these buggers, and spell my name across a few petri dishes.

edit:  Forgive the bright spot from my lamp, it was the only way to get the letters to show.

J

Above you can see the letter “J”.  If you look to the right and bottom of the J you can see clumpy looking gunk – that is egg chunks.

I started out by boiling 300 g of sliced potatoes and 1 raw egg  in ~500 mL distilled water for 30 minutes.  Next I filtered the solution through numerous coffee filters and then filtered the solution a second time through a 0.45 micron membrane.  10 g of sucrose and 10 g of agar-agar (hardening agent) were added and stirred for ~10 minutes.  Next I sterilized the media and some glass petri dishes (~$1.50 per dish) in a pressure cooker for 25 minutes at 15 psi.  After sterilization I poured the media into the dishes and allowed them to cool and harden.

My open dishes

My open dishes

After I finished sterilizing the media I noticed the solution had clumpy-chunky junk floating all about.  I assume this was leftover egg that got through the filters, though it could also have been impurities from the agar-agar (least likely of the two).

I used a cotton swab to collect bacteria from my teeth and gums and I spelled my name (a letter per dish) across the plates using the swab.  On one plate I put nothing and on the last plate I spit into it to cover the whole thing.

e

e

I was happy to see that everything worked perfectly.  The dish with no bacterial additions had no growth.  The dish I spit into had growth all over and the dishes I wrote letters on only grew in the shape of the letter itself!0

s

s

Unfortunately condensation leaked all over the plates when I flipped them and blocked some letters from being photographed.

For future experiments I bought a “fitness multivitamin” that contains all of the amino acids and I am going to try and use it as an egg/tryptone/peptone replacement.

Isolate and drink your DNA with bacardi.

June 28, 2012

DNA tendrils floating between a sandwich of banana puree and booze.

This instructable was based off  a ScienceHackDay SanFrancisco team that I was part of along with Patrik D’haeseleer, Bonnie Barrilleaux, Lily Lew, Joseph Elsbernd, and Michelle Peters.  It is really easy to do and a fun way to incorporate molecular biology into your social life!

http://www.instructables.com/id/DNAquiri-the-delicious-DNA-extraction/?ALLSTEPS

Lab grade vs. home grade electrophoresis buffers

June 8, 2012

(note:  wordpress sucks so it screwed up my images some.)

I wanted to find a cheap way to make electrophoresis buffer from easily accessible ingredients and there were two pieces of knowledge that led me to this experiment.  First I had read about some labs using sodium borate (SB) buffer because it gave great results.  Secondly, I knew that borate and boric acid were easy to find at stores in the forms of roach poison (boric acid) and borax (sodium tetraborate).

Originally I had intended to compare a large number of buffers; TAE, TBE, SB (molecular biology grade), and SB (home grade).  Due to my own personal shortages of lab-grade materials I had reduce down to a comparison between TBE, SB (home grade), and SB with EDTA (home grade).

All gels were 1% agarose.  Gel images were made with Foto/Phoresis I transilluminator and gel images were recorded with an iPhone 3GS camera.

Tris-Borate-EDTA (TBE)

Purchased from the “Online Science Mall” as a 5X concentrate.  Without a doubt this worked better than the buffers I cobbled together.

 

SB Buffer (home-grade)

SB buffer made by adding 1.91g of Sodium borate decahydrate (Borax by 20-mule team) to ~800mL distilled H2O (Target), pH was adjusted with 0.4M Boric Acid (Roach Away by Enoz) and then diluted to final volume with more distilled water.

SB Buffer with EDTA (home-grade)

Image

A 600 mL aliquot of the SB buffer, made previously, had 1.2 mL of 0.5 M EDTA (molecular grade) added to reach 2mM final concentration.

I decided to test the SB buffer with EDTA to see if there were any nuclease related problems occurring.  2/3rds of the way through the project it occurred to me I was using Target brand distilled water instead of nuclease free water when mixing the 2-log DNA.  It just simply slipped my mind.  So this third gel buffer test was done with EDTA and nuclease free water to see if the results would be drastically different or not and they were not.  I believe any differences in the images is due to my lack of a proper gel documenting system.

Given the choice between the three buffers, TBE is the superior.  I do not mean to say that one could not use the SB home grade buffers and get usable results, but rather they just are not as good.  Being that store bought TBE and TAE are not crazy-expensive and buffers can be reused a couple of times, there is not a lot of impetus to use home-grade SB buffers.

My results for the SB buffer tests do not even come close to mirroring the results from the Brody paper I linked earlier, I suspect that my preparation or formulation of SB may be flawed.

While unfortunate that I couldn’t test more buffers, I was happy to figure out that TBE will likely work for my PCR projects in the future and if I am ever mismanage my supply of TBE I now know I can make up some SB buffer to get by.

Democratized science instrumentation

May 30, 2012

Arield Waldman was commissioned to and wrote a guidebook about a bunch of DIY science projects that have gone on.  I was interviewed for background and my gel box was included in the paper!  Really cool!

You can check out the actual paper from a link on her blog.

 http://arielwaldman.com/2012/05/29/democratized-science-instrumentation/

Some preliminary results – electrophoresis buffer comparison

May 12, 2012

I did a quick test of four differnt electrophoresis buffers last night.  The buffers were 1X TAE (Molecular-grade), 1X TBE (Purchased), SB buffer (Home-grade), SB buffer (Molecular-grade).

I will run the test again on a better gel rig (that I dislike using because it uses 3x as much agarose and gel stain as 1 mini gel I used below).

All gels were run at 100V for 1 hour.

Image

SB Home Grade (left).  SB Molecular Grade (center).  1 TBE (right).

Image

1x TAE (left).  1x TBE (right).

You can see that the TAE buffer led to gel deformation whereas TBE did not have that problem.

Image

From left to right.  TAE, SB Home-grade, SB Molecular-grade, TBE.

The two SB buffers were noticeably cooler than the TBE and TAE buffers (27C for SB and 33C for TBE and TAE) and the DNA migrated slower.

I think most of the smearing is due to the high voltage used.  The crappy 2-log ladder (that I have had issues with before) also doesn’t help.

Another problem is my power supply doesn’t handle four simultaneous gels very well, the mA jump up super high in order to generate the needed voltage.  I will be running them individuals on the next test.


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