Archive for the ‘Gels’ Category

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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Comparison of agarose, agar-agar, and cleaned agar-agar for gel electrophoresis of DNA

January 31, 2012

(cross posted here and at Citizen Science Quaterly)

Gel electrophoresis is a core technique used in molecular biology laboratories. The gels used for electrophoresis are almost always agarose which is a polysaccharide purified from agar-agar. Agar-agar is obtained from red algae and contains a variety of impurities along with the agarose. Agar-agar is often used as a substitute for gelatin in vegetarian dishes and is used to make some cool culinary creations. Pure agarose is obtained by separating it from agar-agar. I have heard from variety of sources that agar-agar could be used in place of agarose for gel electrophoresis of DNA. Given that agarose costs around $1.00 per gram and agar-agar costs around $0.05 per gram I thought it was worthwhile to check the efficacy of agar-agar. My results show that agar-agar is not an acceptable substitute for agarose (see the image above).

For more details and thoughts read the rest of this post.

Cleaning the Agar-agar

The night before I ran the experiment I decided that it would be nice if I tried to “clean” the agar-agar of some impurities and run this as an additional treatment group. Since I did this last minute I did not completely think my plan of attack through and actually made the agar-agar worse. I used 50% isopropanol and distilled water to rinse the agar-agar through a coffee filter.

After running the experiment and seeing my results I did the research I should have done prior to the experiment. A quick google search showed me a technique for purifying agarose from agar-agar using propylene glycol. Click here for the site in question.

Failed Purification Protocol:

  1. Weighed out 11.7g agar-agar into a coffee filter (2 thick).
  2. Saturated agar with distilled H2O (dH2O).
  3. Poured 100mL of dH2O through the slurry and allowed it to drain.
  4. Stirred slurry with a spatula.
  5. Pourd 100mL of dH2O through the slurry and allowed it to drain.
  6. Stirred slurry with a spatula.
  7. Poured 100mL of 50% isoproanol into the slurry.
  8. Stirred slurry with a spatula.
  9. Once the majority of fluid drained out I placed the agar-agar and coffee filter into an oven at 90C for 5 hours to dry.

Experiment Parameters

  • 1.2% gel
  • TAE Buffer
  • 2.5 hours
  • 70 volts
  • Biotium GelRed Stain (used as a precast additive)
  • Promega 1kb DNA Ladder (left lane)
  • NEB 2-Log DNA Ladder (right lane)

Pictures and Comments

Agarose gel atop the UV transilluminator.

I had minor issues taking pictures of the gels because too much light was reaching the camera lens so I made a aluminum foil frame to block out extra light.

Close-up of the agarose gel’s bands (leftside = (+) pole, rightside = (-))

All 3 gels deformed on the end closest to the (-) electrode and I am not sure why. I suspected temperature at first but it subjectively seemed constant throughout the gel rig. Next I thought it was the gels positioning, however when checked I found each end to be equally distant from both electrodes.

Compare the picture above to the reference gel below (in black and white). This reference gel was the first gel I ran with my gel rig and it ran for 80min at 70V using the same TAE buffer used in this experiment and there was no deformation of the gels wells. The two differences between the reference gel and the newest gels were time and staining compound (2.5 hours vs. 80min and ethidium bromide vs. GelRed). If this problem persist I will have to run a bunch of gels to figure this out.

See blurb above for information about this reference gel.

All 3 experimental gels on the UV transilluminator. From left to right; agar-agar, cleaned agar-agar, and agarose.

Conclusions

Based on my results I do not recommend purchasing agar-agar for gel electrophoresis. The only exception I would make is if the intent is to use propylene glycol to separate the agarose from the agar-agar (click here for agarose separation with propylene glycol). Keep in mind that there could be nuclease and cation contamination in the propylene glycol purified agarose.

While agarose is a lot more expensive than agar-agar, it is not expensive relative to everything else needed for conducting molecular biology at home. For the sake of simplicity, reliability, and time I intend on using only agarose going forward.

What is next

My next post will compare running buffers between gels – specifically molecular biology grade Tris-Acetate-EDTA (TAE buffer), Molecular Bio grade sodium borate buffer (SB Buffer), and sodium borate buffer made from borax and cockroach poison (boric acid). If there is any simple household substances you want me to try as a running buffer, let me know. I have a lot of school and work projects going on so this upcoming post may be a few weeks out.

Finally got around to testing some gel electrophoresis.

January 16, 2012

It took much longer for me to get a round to testing out my gel electrophoresis equipment than I thought it would.  For now I have merely got it to work.  Next I will try and fine tune it to increase the quality of the gels.  More on that below.

This isn’t the most informative post but I was kind of frustrated by a lack of information when I was troubleshooting so I figured I would throw some data out and hope it helps someone.

Note:  The cameras on my phone and iPad both captured more wavelengths of light than I could see, so these images look worse than the gel actually is.

Zoomed out gel.

Zoomed out gel.

Gel closeup

Gel closeup

Gel Parameters:

  • GEL = 1.5% food-grade agar-agar gel (not agarose)
  • DNA LADDER= New England Biolabs 2-log DNA ladder
  • STAIN = GelRed Stain (Vendor; Biotium) (Approx. Equiv. to Ethidium Bromide, except safe).  Stain was used in the precast gel (1x) context.
  • TRANSILLUMINATOR = Fotophoresis I (Fotodyne)
  • BUFFER = TAE (MB grade reagents)
Failed gel.

Failed gel.

The image above is what the first two gels looked like – no fluorescence at all.  I still do not know for sure why they failed but I narrowed it down to either the composition of the DNA ladder or the staining method.

My set-up worked when I used GelRed in the molten agar-agar and composed the DNA ladder per the manufacturers instructions.  I used 5-10uL of ladder and 1-2uL of loading buffer on the failed gels whereas I used 1uL of ladder, 4uL H2O, and 1uL loading buffer on the successful gel.  During the failed gels I tried to use the 3x post-electrophoresis stain procedure with staining times between 0.5-1.5 hours – all with no luck.  Obviously changing two variables at once confounds the results – but at least I have a baseline now.

The setup

The setup

This was the gel box I built and used.  You can read my instructions for how to build it here.

Muh lab.

Muh lab.

This is ~90% of my apartment lab.

My next goal is to work out how to fine tune the procedure.  I am going to compare the gel quality in cases where I use reagent grade vs. industrial and food grade chemicals.  I am hoping that borax (sodium tetraborate) and roach poison (boric acid) buffer will work as good as its reagent grade cousin.

Update: Sterling silver as an electrode

January 14, 2012

Sterling silver wire does not work as a cathode or anode for gel electrophoresis.

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, as finished as it is going to get.

October 2, 2011

I kinda-sort finally finished the LEGO electrophoresis box.  Saying  ‘finished’ is a half-truth of sorts because I decided to abandon the nearly functional gel box.  Everything worked out but a few issues made the LEGO gel box more effort than it was worth. Keep reading for the details.

Final box

In the beginning….

When I originally set my mind to making the LEGO gel box I decided to test whether it was true that acetone would melt the ABS plastic (which the LEGO’s are made of) and thus fuse the bricks together and I wanted to test how nice of a gel could be cast.  The image below shows a still molten agarose gel being cast inside the rotatable gel mold.  To my delight, it was true that acetone would meld LEGO’s together and the LEGO’s worked great for casting a gel.

The prototype

I decided to class things up a bit and make the second version of the gel box all clean and spiffy looking (see image below).  The main impetus for using a single color LEGO brick was to avoid the color smearing that was inevitable.  The application of acetone melts and leads to smearing of the different colors.  In short, it looks dirty.

Black is the new black

Some assembly required….

The gel box can be assembled in an infinite number of ways and each person can go about things however they want.  The box I built was 12 LEGO pips by 28 pips and was 4 bricks tall.  Being the second attempt at a LEGO box I decided to step things up a noth and add 4 small protrusions on the inside of the box to hold the gel mold in place.  These protrusions stick out one LEGO pip and they do not obfuscate the electric field as the gel mold already blocks this area.

I entertained quite a few electrode placement variations before ultimately coming up with the design you see above.  I originality wanted the cables to plug into the side of the box, but the size of the banana plugs and the stubbornness of LEGO bricks to be drilled neatly prevented a side mount.  The LEGO plates I ended up using rest on top of the gel box and hang over it.  Take a look at the image below.

No electrodes

The image above is the gel box without electrode flaps and the image below is the box with electrode flapss.

With electrodes

Creating the electrode holders was easy.  Start with a LEGO plate that spans across the gel box and decide where to place the hole.  Once the spot has been chosen, drill a hole by starting with a small drill bit and moving up sizes until the hole is the needed size.  Starting out at the largest bore size will likely cause the plastic to crack or warp.

My reaction to drilling LEGO’s surprised me. -I was filled with a combination of childhood nostalgia and irrational love for LEGO which urged me to quit!   I nevertheless worked past my irrational love and generated the part you see below.

Drilled hole.

The next step is to attach the bannana jack.  The jack I bought attached to the LEGO plate by a screw on the underside (no gluing required).

Banana jack.

Electrodes attach to the jack similar to how the jack attached to the LEGO plate – via a screw pinching the material.  To make the electrode I stripped some wire and twisted it to form a cable.

Electrode

Once the electrodes are set-up its time to work on some finishing touches.  First, a gel tray or cast is needed.  The image below is the prototype tray I made, I chose this image over the new one I made because it is easier to see this one (on account of my camera being unable to image the black colors).  The mold is  a few LEGO plates, flanked on two sides by 1 pip wide bricks and covered with smooth plates.  The molds I built were 8 pips by 8 pips.

Mold

Building a lid for the gel box is critical, that is unless being electrocuted sounds appealing (note, the electricity could kill someone).  I lacked any single LEGO plate that could cover the entire span so I made my lid two LEGO plates thick and staggered multiply pieces so I could span the gap.  I lined the top of the gel box with smooth-topped plates and a couple normal plates so that the lid would attach but be easily removable.

Final box

After the box is assembled it is time to  weld all of the bricks.  When I chemically treated the gel box I used pure acetone and wiped all sides of the gel box.  I avoided welding the lid because there was no need to make it waterproof.

As an aside I recently read an article from MAKE magazine that gave a suggestion for making ABS glue.  They suggested adding ABS plastic flakes or pieces to a small bottle of acetone and letting the plastic dissolve over night.  The addition of plastics supposedly helps with the combining of the ABS plastic later on.  I have never tried this, but I will if I ever need to chemically weld plastics.

After acetone was dried up I performed a water test.  To my delight, the box held in water almost perfectly.  There were only three leaks present and all of them were at the junction between the walls of the box and the yellow base-plate.  I decided to be a cheapass and not purchase silicone to seal the bottom edge and I now regret this.  Instead of the silicone, I used a thick bead of PVC glue all around the bottom.  This was a bad idea.  The PVC blocked up two of the holes just fine, but the third was not stopped.  The problem was not the PVC glue sucked at sealing the bottom but that I could not cut out the PVC glue and re-seal the problematic spots (PVC glue melds with plastic).  When I tested the box after PVC glue treatment, the remaining leak was worse than the three original leaks, combined.  The problem was damage from transportation.  Going from my apartment to the lab led to the base-plate becoming even more loose and made the leaking worse.

The leak was the straw the broke the camels back and made me abandon the project. In my opinion,  if moving the gel box around could easily lead to leaks forming then the box was not worth the effort and I should probably just use a Rubber-Maid container or something.

The last problem I had was with chemistry.

The image below shows a electrochemical reaction that, while kind of interesting, was frustrating to encounter.  The twisted wire worked just fine for the anode, but the cathode  reacted to the current and chemicals.  I have no clue what exact reaction is occurring but the end result was the wire becoming a striped red and green color (which I think means the wire is made up of separate smaller wires wound together, probably copper and something else).

Frustrating reactions

(-)

All done.

The following two images are of the (mostly) finished gel box.  Despite the issues I had the box worked and so I am calling this a success even though I abandoned the project at the finish line.

In operation

 

Hooked up and with a lid

While I may have given up on using LEGO’s I have no given up on building a cheap box (<$10 a box).  Below is a preview of the new gel box I am working on and I will blog about it later.

Cheapo box

 

 

 

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.

 

Save money on electrophoresis hardware and chemicals.

August 18, 2011
Casting a gel.

Casting the gel took longer than running it.

Gel electrophoresis is a mainstay of molecular biology.  So obviously making it cheaper will benefit both research labs and DIY biologists alike.  While perusing blogs and other websites I collected a number of projects and instructions that ought to help make one a cheapass.

Sodium boric acid buffer (cheaper faster gels)

The running buffers TAE (TRIS acetate EDTA) and TBE (TRIS borate EDTA) are two of the most common types of electrophoresis buffers.  According to a paper by Jonathan Brody and Scott Kern (link below), a sodium borate (SB) buffer is a better choice than either TAE and TBE.  At normal electrophoresis voltages their results suggest the 3 buffers (TAE, TBE, SB) are roughly equivalent, with a potential edge given to SB buffer.  However, in the high voltage contest (300V+ ish) SB is the better choice (TBE and TAE melt at high voltages).

The SB buffer utterly dominates TBE and TAE for reasons I will explain.  SB buffer is cheaper per gel than the other two – TAE=$0.27, TBE=$0.67, SB=$0.07 (data from the Bordy et. al. paper).  The results quality of a gel in SB buffer at normal voltages and high voltages is as good as the other two buffers at normal voltages.  A higher voltage leads to faster migration time and the paper and comments on the paper suggest a 60 minute run in TAE or TBE can be completed in 20 minutes with SB buffer.

I tried to make some SB myself but had some issues, I think, because I lacked boric acid (to to adjust the pH).  I tried making the SB buffer with sodium tetraborate and 1N HCl but the gel melted at 300V and 150V.  I suspect part of the issue is the chloride ions from the HCl and the other part is the power-supply.  I am using a 75W max unit and repeatedly the machine hit max power and the voltage couldn’t be maintained.  Once the boric acid arrives I’ll be trying the experiment again.

Recipe (from Brody’s paper)

    • 1X Stock
      • 5mM sodium tetraborate decahydrate (disodium borate decahydrate) adjusted to pH 8.5 with boric acid.
    • 20X stock
      • 100mM sodium tetraborate decahydrate (disodium borate decahydrate) adjusted to pH 8.0 with boric acid (when the stock is diluted to 1x the pH will be correct)

Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis (PDF, Minnesota State University)

DIY Hardware Guides

I am less familiar with the hardware side of electrophoresis so I will simply link any readers over to the following projects.  When I get the time to build one I will share my experience.

Build a gel box – University of Utah’s Instructions (PDF)

Open Gel Box 2.0

Drinking starw electrophoresis 

Electrophoresis and DNA extraction from common items.

Staining trays.

March 9, 2011

This kind of boggles my mind, why would anyone spend $45 dollars on a plastic tray for gel staining?  The mere fact that a company even made this product makes me think that either I am too dumb to understand the use of these things or there is a hell of a lot of dumb and/or apathetic(as in they are not spending their own money on and “just don’t give a fuck”) people out there.  A tray for gel staining is such a simple thing that there is going to be boundless alternatives out there so I am only going to list a couple to get your wheels spinning.

Gladware Containers

Strangely enough, Gladware has many of the selling points of the $45 tray I mentioned earlier – amazing features such as “Shake Usable” and “Stackable” – but for a fraction of the cost ($3-4).  I have seen these used in a lot of teaching and research labs and they rock because they are lightweight, reusable, dirt cheap, and easily available.  There one drawback that I have seen is that they look really ghetto and cheap – but Science isn’t about the glamour right?

 

Pipette Box Lids

Ignore the box and check out that sexy lid.

Any lab staining gels is going to be using boxes upon boxes of pipette tips (unless you reload them manually or use the tray inserts…) and so you’ll inevitably end up with lots of waste.  Why not turn that waste to good use?  Pipette box lids meet the positively stringent criteria for staining gels – deep enough to hold a bit of fluid a gel slab.  I like these lids better than Gladware for a number of reasons.  First the box lids, while still ghetto, do not look nearly as ghetto and could pass as something quite acceptable.  Second, pipette boxes are made from plastics that can withstand autoclaves(typically atleast) and so this fact suggests that these lids can tolerate some microwave related abuse.  Finally and most importantly – these are likely already laying around the lab and so there is no need to travel to a store or order anything.

99 Cent Stores

Depending on your region there may be some discount $0.98, $0.99, or Dollar-Tree stores around and if so they are worthwhile to scope out.  Most of these stores carry quite the eclectic selection of glass and plastic kitchen ware and there may be some vessels that could serve as trays.  Just remember that the selection at these stores is hyper variable like certain immunoglobulin genes I know, so should you find some trays you like buy all that are available.