Posts Tagged ‘tbe’

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)

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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.

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.

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

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1x TAE (left).  1x TBE (right).

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

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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.