Finally got around to testing some gel electrophoresis.

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.

Advertisements

Tags: , , , , , , , , , ,

2 Responses to “Finally got around to testing some gel electrophoresis.”

  1. Los Angeles Precast Says:

    Los Angeles Precast…

    […]Finally got around to testing some gel electrophoresis. « Cheapass Science[…]…

  2. samples Says:

    Thanks for a marvelous posting! I definitely enjoyed reading it, you happen to be a great
    author.I will make certain to bookmark your blog and will eventually
    come back later in life. I want to encourage you to ultimately continue your great job, have a nice evening!

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s


%d bloggers like this: