Category Archives: KochLab Stuff

Lab Contamination!!!

This morning while setting up for a time trial experiment, I noticed the 20% yeast sample didn’t smell like yeast anymore. It smelled like a mixture of yeast and something else. So I setup the experiment, took initial measurements, and then analyzed the sample in the microscope. This is what I saw:

Surrounding my slightly modified yeast are these tiny things, that look like super small e. coli so they are probably some bacteria or perhaps they are some kind of spore. Regardless that was not at all in the sample from yesterday (see above), and looks nothing like the e. coli that I temporarily believed was adapted yeast.

So I began a mission to decontaminate the lab. After the cleaning I just did, nothing is alive! Not even myself! In fact I’m not even writing this… (Note to dead future self: Sorry :-\)

Anyways, I began by bleaching the fuck out of everything. The incubator got it the worst as I basically drowned it in bleach. I scrubbed real hard with this super awesome huge bristle brush. I let the bleach sit for about 10 minutes and then wiped it down with clean rags.

Next I used the Activeion Ionator EXP, which was loaned to me by the custodial staff here at CHTM. It’s a spray that ionizes water to clean and disinfect, and supposedly can kill viruses and bacteria. After the bleach treatment on the incubator, I Ionated it and wiped it down and allowed it to air dry.

Then I used the Ionator EXP to clean all the bench tops and all my lab equipment (pipetters, racks, scales, hot plate, etc). I finished by emptying my current supply of YPD and made new stocks for use tomorrow. I feel sad that I had to throw about $80 worth of D2O down the drain, but I gotta be careful in the lab and so it had to go.

Tomorrow I will start the D2O adaptation experiment again (Round 3!) and let’s hope the contamination issues are behind me.

 

All #SciFund Expenditures

I’ve been posting my expenditures as I buy them, but I thought it would be beneficial to everyone (myself included) to document the total spent. So, here it is for your viewing pleasure:

If you have any questions about any of the line items, ask in the comments!

#SciFund Expenditure!

Today I bought some goodies to help me analyze and control my experiments:

  1. light timer to control the lights for the plants. Over the thanksgiving break I was unable to come into the lab and since I doubt anyone else was in here, I’m sure the plants saw no light for 4 days. This will remedy that!
  2. A Macro/wide angle lens.
  3. A zoom lens and tripod.
  4. A microscope objective.
  5. Deuterium depleted water (from Sigma).

I bought the iPhone accessories to make my data collection process more efficient. Since they were relatively inexpensive it wasn’t a big gamble.

Microscope Pixel Calibration

It is important to provide scale when showing microscope images. Personally I’m not a huge fan of scale bars and would prefer to know the dimensions of an image and the conversion factor. For our camera/microscope at 10x magnification it’s almost exactly 1um/px. This calibration was performed by Andy Maloney using a 60x objective. See below for details.

Without power part 2

For the second day in a row, the power is out. This time the maintenance guys are trying to find the cause and fix the issue. Hopefully we get it back soon. The positive is that I got power to the -20 and -80 freezers, the 4C fridge, and the incubator so my experiments live on!

I’ll update when I know more.

Power is Restored

Apparently the circuits labeled surge protector had been knocked out and the areas of the lab that had no power were connected to that circuit. Power to all areas of the lab are now restored. Yay!

Time to make the donuts…

Lab Power Outage

At some point over the weekend the power at CHTM went out. Half the circuits in our lab are down as a result. I have zero power to half the wetlab station, every circuit in the front of the lab (where the balance, pcr machines, centrifuge, ph meter, and computer are kept), and also the incubator shaker.

Hopefully this doesn’t set me back too far.

 

Glycerol Stock Creation

I made glycerol stocks of the yeast that started the entire D2O adaptation experiment, and now that I may have a D2O adapted strain I made another glycerol stock for safeguarding. I used the protocol found here for reference, with the exception that I doubled the recipe.

Shotgun DNA Mapping Buffers

I worked with Pranav to make some H2O and D2O buffers for the shotgun DNA mapping experiment. We use a buffer that we call Popping Buffer for our tethering experiment. The name comes from the fact that this buffer was used during Koch’s (and potentially others’) experiments that involved “popping” bound proteins off DNA while unzipping. The buffer is (final concentration):

  • 50mM NaCl
  • 50mM Sodium Phosphate (which is a mixture of dibasic and monobasic sodium phosphate)
  • 10mM EDTA
  • 0.02% Tween-20

We made two 100ml amounts of solution in D2O and H2O respectively. And the buffers were concocted from separately made solutions in each water type:

  • 4M NaCl was made as a solution in both D2O and H2O
  • 500mM Sodium Phosphate (monobasic) in D2O and H2O
  • 500mM Sodium Phosphate (dibasic) in D2O and H2O
  • 100mM EDTA in D2O and H2O

From some Popping Buffer we made BGB (Blotting Grade Blocker) in both D2O and H2O at a concentration of 5mg/ml (about 15ml volume). And we also made aliquots of anti-dig in PBS (from only H2O, since we aliquot 20ul amounts and then add 180ul of Popping buffer when it is experiment time).