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Author Topic: Strawberry and Banana DNA extraction and visualization  (Read 653 times)

da3v

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Strawberry and Banana DNA extraction and visualization
« on: August 23, 2015, 04:43:28 PM »

Partially MacGyver protocol, partially Edvotek.


http://www.scq.ubc.ca/the-macgyver-project-genomic-dna-extraction-and-gel-electrophoresis-experiments-using-everyday-materials/


http://www2.ca.uky.edu/brei/Teach/4-H/Info/Macgyver%20Electrophoresis.htm


http://wikieducator.org/Lab:_DNA_gel_electrophoresis


We extracted DNA as follows:
Squish a few pieces of fruit (a few frozen strawberries or a piece of bananna) in a ziploc baggie with:
   some Edvotek buffer (I need to get details)
   a squirt of dish detergent (I need to get details)
   a few drops of contact lens solution

We then gravity filtered the resuting mush through coffee filters in plastic cups. Some of the filters broke. In retrospect, an aeropress might have been a really great or a really stupid idea.
We then took the resulting fluid and suspended a layer of 0degreeC alcohol above it in a small clear disposable plastic shot glass.
We swirled the interface of the two liquids with with various stir-sticks and collected the hazy fibrous goo ("DNA Snot" as it is described in the protocol) into microcentrifuge tubes. The stir-stick that worked the best was a short piece of 1.75mm 3D printer filament with the end bent in a slight L or J "hook".
We centrifuged the the tubes at "15" (15K RPM?) for about 20 min, poured off the supernant, rinsed with buffer, hand vortexed, centrifuged again, poured off supernant, rinsed with buffer, hand vortexed, and then left to "disolve into solution" for a week or two. (2 weeks for the banana goo, which was dark brown/gray in color, approx 5 days for the strawberry goo which was more translucent-whiteish in color).


We prepared "MacGyver" and Edvotek loading buffer as follows:
  (MikeL can chime in here, or I will check notes the next time I am at the lab)


We prepared Edvotek gel using .39g of agarose, 1.0ml of 50x concentrated buffer, and 49ml distilled water. Microwaved for 45 seconds, let cool to 140 degrees F (measured with stainless meat/coffee thermometer) poured a "deep gel" with very little liquid left over.


After the gel had set thoroughly, we placed in buffer, added buffer to 3mm+ above the surface, and then took turns pipetting the DNA+loading buffer Alternating banana and strawberry (alternating Red (MacGyver) and Blue (Edvotek) buffers) :
Bananna-Red, Bananna-Blue, Strawberry-Red, Strawberry-Blue, etc.


We set pipette to approximately 100ul, and could have used less.


We ran the gel for approximately an hour (need to look at timestamps) until the loading dye was almost to the right of the gel. The red dye was visible, the blue dye was not. (Score +1 for MacGyver)


We stained using Edvotek "Flash Blue" for 4 minutes, 30 seconds, then destained in 75ml distilled water for about an hour. (Save stain in sealed container for future use)
http://www.edvotek.com/site/pdf/DNA_Stain_Guide.pdf
Then replaced water with 75ml fresh distilled, and destained for several more hours.




We could definitely see a difference in the banding of the strawberry vs the banana. We did not see a difference in the end result due to the loading dye used, but the Red was a LOT easier to work with.


Pictures at:
https://plus.google.com/+DaveCaseyin3D/posts/3y6dXzgmySJ?utm_source=chrome_ntp_icon&utm_medium=chrome_app&utm_campaign=chrome&pid=6186291217162643986&oid=111400474599592670239


and


https://plus.google.com/+DaveCaseyin3D/posts/7XEmbs2o8UJ?pid=6186292664750480610&oid=111400474599592670239




Things we learned:
  "J" shaped 3D printer filament works pretty well for gathering extracted DNA
  Using a slide viewer light table under the gel box made loading the dye+DNA significantly easier to see.
  Flash blue stain can be saved and reused.


Things that could have gone better:
  Keep better track of time.
  Watch the Edvotek videos
  I tore the gel while transferring it to a ziplock bag after de-staining.


Pipetting errors we encountered, and might want to practice M&M protocol to correct?
  poking through bottom of well, with loading dye+dna going under gel. see "smudged corner" in picture
  squeezing out additional buffer after lifting out of well, but before lifting out of tray (loading dye on top of gel)
  lifting up on plunger before pulling out of well. (Sucking some of the dye back into pipette
  dealing with bubbles in tip of pipette


Wondering about a quick pipetting exercise involving dye and specific volumes for practice.


Questions that still need to be answered:
  Why were the tracks darker along the outer edges and lighter in the middle?
  We did see a difference between strawberry and bananna, what was it that we were we looking at?
  We hand long tracks, not narrow bands. Why?
  What kind of buffer did we use? What should we have used? See: https://cheapassscience.wordpress.com/2012/06/08/lab-grade-vs-home-grade-electrophoresis-buffers/
  How should we dispose of blue destaining water?




All in all, a pretty satisfying hello world, thanks to everyone who showed up and helped work through it!

darkmoonsinger

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Re: Strawberry and Banana DNA extraction and visualization
« Reply #1 on: August 24, 2015, 09:21:04 AM »
As you go along, would you consider blog posting some of your adventures? This is pretty nifty stuff.

willasaywhat

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Re: Strawberry and Banana DNA extraction and visualization
« Reply #2 on: August 24, 2015, 09:43:44 AM »
All we need are some pics to go with it and I can throw together a post. :D

cosmicaug

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Re: Strawberry and Banana DNA extraction and visualization
« Reply #3 on: August 24, 2015, 06:06:02 PM »
What kind of buffer did we use? What should we have used? See: https://cheapassscience.wordpress.com/2012/06/08/lab-grade-vs-home-grade-electrophoresis-buffers/

Nice find! Or maybe it just seems like a nice find to me because I've been out of the lab for so long. All I know is that I had not heard of sodium borate buffer and that what we used was 0.5X TBE. That seemed funny to me given that 1X is meant to indicate the full concentration but I am guessing that running with the concentration diluted to 1/2 "normal" concentration reduces problems with gels overheating by reducing ionic strength? In any case, it seems to be a pretty standard way of running agarose gels.

The sodium borate buffer seems to be nothing more than 0.5X TBE without the T (Tris) and the E (EDTA). It's surprising that so many gels have been run with the TBE version when the tris is unnecessary  (if the Brody & Kern paper is correct).

But Joseph Elsbernd at the Cheapass Science blog post you linked to sure didn't like his results. I wonder what went wrong with that?

Lensman

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Re: Strawberry and Banana DNA extraction and visualization
« Reply #4 on: August 25, 2015, 11:20:09 AM »
Thanks for the experiment report!  Cool things are happening at the lab, we just need to get the word out!