User:Hetmann/Notebook/2007-2-16

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91r Second Semester

Whoohoo lab time~!

Clean-Up

Many of the plates from the summer needed to be tossed out (plates last only about 1 month), and since we have all of the important plasmids, I threw away some of cyano's plates. I got a little carried away, and expunged the Thing Formally Known as the Cyano Plate Refrigerator of all non-cyano plates that were over a month old. I will confirm with the Pre-med Formally Known as Zhipeng Sun that we do, indeed, have all of the crucial BioBricked plasmids before tossing out the rest.

Additionally, I emptied some PCR tubes with arcane symbols on them from the Cyano Freezer, and in general organized everything (somewhat).

Western Blot

I completed 1/2 of my first Western Blot (of many) this semester.

There are slight adjustments to the protocol and they will be outlined below.

Foreward: before doing anything productive, I did something counter-productive. Unbeknownst to me, I dropped two samples from the oscillation experiment on the floor Wednesday, and recovered them 22 hours later on Thursday. These samples have "X"'s on them, to indicate Cyano-sadness.

More forewardness: All of my activites were completed in room 5096.

First, I performed a 1:10 dilution of the pelleted cells from the time point data collection series (the oscillation experiment). I believe ZZPS reconstituted the pellets initially in around 150ul of PBS, and I took 10ul of each samples and reconstituted them in 90ul of TBS. These samples are in the Cyano Freezer (also known as the entire mcb100r freezer, also known as WWII.5).

Second, I added:

  • 5ul TBS/sample
  • 4ul 5x Sample Buffer
  • 11ul ddH2O

...to PCR tubes. BAD IDEA!

Third, I added these time PCR tubes to the PCR circular container and put them in the boiling bath of water on the heater. These tubes were supposed to be boiled for 2 minutes, but I spent a little bit of time fishing out the tubes, which, due to the force of the bubbles, had bumped out the tubes. In the future, use 1.5ml tubes.

Next, the tubes were placed in the Thermo centrifuge next to the gel-imaging computer, since these is the only centrifuge that contains holes small enough for the PCR tubes. Run @ 13,000 rpm for 1 minute. (Ideally, these tubes should be instantly inserted into the gel at this point, but they were not.)

Running the gel: I used a 12% Tris-Glycine Gel 1.0mm X 12 well gel (which ran out today).

The gel apparatus has 4 moving parts, excluding the acrylamide gel.

The acrylamide gel should be opened over the sink. After opening the gel, great care should be taken so as to not destroy the lanes (be very careful with the comb). Use a syringe to clean out each of the rows, and then insert the gel into the gel apparatus.

To load the samples, the long pipet tips are used. Hold the pipet tips in the solution for an extra 5 seconds so that it collects as much solution as possible.

Ladder: SeeBlue(R)Plus 2 Prestained Standard (1x)

For amounts <10ul: use p20

For amounts >10ul: use p100

Note: a mistake was made when loading the gel. Some of the lanes have less amounts of sample + solvent than others. This is because of the bubbles that were ejected from the pipet tips. If there is a problem with regards to the amount of protein in each of the lanes, use 10ul or be extra careful with the pipet tips.

The acrylamide gel was run for ~1:50 @ 140V, but it should have been somewhere around 1:30. I was making the transfer buffer during that time, and did not get to the gel until later.

Making TrisGlycine Transfer Buffer for Semi-dry blot:

Add 80 ml Tris-Glycine Transfer Buffer (25X), 100 ml Methanol, and 820 ml ddH2O

This should be somewhere around 1 liter of Transfer buffer.

Completion of gel: to be continued