Posts Tagged ‘science’

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.

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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.

How to build a ghetto micro-environmental chamber for inverted fluorescence microscopy.

September 8, 2011

This project is very easy, super cheap, and has a very specific application.

Background

Our lab has been trying to figure out ways to cheaply set-up an environmental chamber for time lapse imaging of tissue cultures.  Environmental chambers are typically expensive or just a pain to make and so I threw this ghetto chamber together.  This chamber works for us because we use an inverted confocal microscope.  This make-shift chamber is opaque and will not work for any set-up where light passes through the bottom of a sample and out the top (in our case light passes up through the bottom and then is scattered back down).

The chamber requires an appropriate gas supply (our goal is to use air with 5% CO2) and some way to regulate the flow to the chamber.  We are having trouble locating a good gas supply so the chamber hasn’t been tested out yet.

 

What you will need

    • Equipment
      • drill
      • drill bit
    • Reagents and consumables
      • a pipette box lid that is larger than culture plates
      • barbed fitting with threaded end
      • tubing (runs from fitting to tank)
      • washer
      • nut

 

Required consumables.

Below is what the inside of the pipette box lid looks like.  This is also the orientation the lid should be in when it is drilled.

Pipette tip box lid (bottom view)

Drill a pilot hole (use a smaller bit than your final size and if needed increase from one size bit to the next until the final bore size is reached).  The pilot hole is important because pipette tip box lids are likely to crack when being drilled.

Pipette tip box lid with drilled hole

Now the chamber can be assembled.  Push the threaded end of the barbed fitting into the chamber.  Place a washer and then a nut onto the portion of the threaded barb that is on the inside of the chamber.  The figures below shows the finished chamber on the outside and inside.

Micro-environment chamber assembled.

Inside of the micro-environment chamber.

Naturally a longer tube than the one I have depicted above will need to be used to reach the air tank.  The short piece I have attached is there for display.

DIY centrifuges, used centrifuges, and a fun experiment.

September 3, 2011

The background that led to the experiment

I had been thinking about building a centrifuge out of a blender but first I decided to research what other people had done before I tried my own hand at it.  I ran across a number of designs which included  “Dremmelfuge“” and a handheld centrifuge which will eventually damage something.  The best of centrifuges was one that used mixing bowls and a blender.

My own thought was to attach the caps of dry-erase board markers to a blender rotor using wire and gorilla glue.  In my design I wanted to use the blender pitcher as a safety vessel for in the likely event something went flying – but this provided a lot of design challenges.  Ultimately I decided that my idea was bad and most of the other designs were too risky.  If I were to ever revisit centrifuge construction definitely would make a variant of the blender which used mixing bowls.

All of this ended with me surfing eBay and discovering it was not too expensive to just buy a used centrifuge (from $100-150) – which I did.  I manged to find the centrifuge depicted below.  I find the aesthetic design to be quite pleasing and it is a bit sad that this style is not used anymore.  Now that I have a centrifuge, it is time to use it (see below for more)!

My little buddy. Cheap and effective.

A quick centrifuge explanation

Centrifuges spin samples around extremely fast (often >10,000 RPM).  Doing so causes centripetal force to be exerted upon the samples.  In the case of my centrifuge, it spins fast enough for the samples to have the force of 13000 times earths gravity exerted upon them.

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What happens when ketchup, milk, sriracha sauce, and russian salad dressing are exposed to 13,000 x earth gravity for 30 minutes?

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The source material precentrifugation.

Here are the specimens, precentrifugation.  From left to right we have non-fat milk (Trade Joe’s), Ketchup (Heinz), Sriracha Sauce (Huy Fong Foods) and Russian Saland Dressing (Wish Bone).

First we load the machine…

Rotor with tubes.

Next we run the machine for 30 minutes and we get….

sriracha sauce 13000 x g 30min

Separated out kind of like blood  (serum on top, cells on the bottom).  I would guess that the bottom layer is chili pepper fragments.

russian salad dressing 13000g 30min

This salad dressing has A LOT of ingredients and I will even try to guess as to which layer is what.

nonfat milk 13000g 30min

Milk proteins should be what has collected at the bottom.

heinz ketchup 13000g 30min

The ketchup surprisingly did not separate into layers.

My take home message from this experience was that sometimes the effort and danger in building something myself may not be worth it when one considers the cost and value of used equipment.