Posts Tagged ‘gel’

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.

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How to build a $21 gel box.

October 22, 2011
My box

My box

My results

Citizen Science Quarterly asked me if I would like to blog at their website and I said sure.  The first thing I blogged about was how to build a gel box for 21 bucks.

Click here to read about it.

LEGO electrophoresis box update.

September 19, 2011

I spent some time this weekend on improving the LEGO electrophoresis box.  Below are two images.  I should be done with the whole thing and testing it some time this week.

One thing holding me back is that I have been having a lot of problems soldering cables.  I have never soldered before and I think the iron I bought is defective.  Eventually I will work my way through the problem.

Box with cover.

Box without cover

LEGO electrophoresis box and gel cast (prototype)

September 11, 2011
Completed casted gel

(visit the photo gallery for more pictures)

Gel boxes are ridiculously expensive, so much so that  I was able to buy a used electrophoresis machine off eBay for less than the cost of a gel box!
My first attempts in building a gel box involved scoring and sawing 1/4″ plastic boards in an attempt to cut square pieces for a gel box.  This method was too crude and I could not get a straight  line.  The only positive thing from this attempt was I found that PVC cement was able to join plastic pieces together to make a strong water-tight seal.
While brainstorming I reckoned  that LEGO’s would be perfect for a gel box.  After googling for a bit I found only one reference to LEGO’s and electrophoresis and it was at the journal of BioTechniques (link here).  It was frustrating to discovery that no digital copy of the article exists and so I decided I would fumble around in the dark on my own.

Casting a gel with LEGOs

The image above shows my prototype gel box and gel mold (with molten agarose).  The box itself is just a frame.  The gel mold is square so that the mold can be rotated once the agarose cools to make for easy gel casting.  The bottom of the mold is covered in those smooth LEGO planks without rivits (I suspect it may have worked even with the rivits, but the acetone treatment would be more difficult).

One of the big design challenges with using LEGO’s is waterproofing the construct.  LEGO’s are made from ABS plastic and from all of the reading about 3D printers I have done, I knew that acetone melts and melds ABS plastic together.  After constructing my gel mold I dipped a Kim-Wipe (lint free paper towel) into some acetone and rubbed it all over the sides and surfaces of the LEGO mold.  Immediately the colors began to smear and the cracks and grooves filled in.  Within minutes the whole thing was dry and ready for casting a gel.
Nearly all of the agarose stayed inside the mold (I added 40mL of 2% agarose) and the mold and gel both were removed and rotated nicely!
I ran into one issue.  The acetone was not completely dry and it seeped up from the base plate of LEGO’s and made a weird tiny plastic ridge (which I later removed with more acetone).  I only gave the LEGO’s about 5 minutes to dry so I am not surprised.  But what was surprising was the patriotic shape theplastic made in my gel!

Flag? Gel?

I am definitely going to pursue the LEGO gel box further and will post more if I have success.