Jim's Sadness Journal

List of Things That, By Not Knowing, Have Ruined Experiments

Contents

1. Labeling Tubes
2. NEBNow Freezer
3. Agarose Gels
4. Protein purification
5. PCR
6. pH Strips
7. FPLC
8. Worldview

1. Labeling Tubes

• I had ten centrifuge tubes labeled 1 through 10 on the top. I forgot to put a line below the digit of tubes 6 and 9 and they were then indistinguishable.

2. NEBNow Freezer

• The inventory understates what is in the freezer. Even if it claims it’s out of something, look through the shelves anyway, it’s probably there.

3. Agarose Gels

• Some DNA loading dyes can be excited by UV light, making it appear like you have a massive blob of very small DNA. This specifically happened with commercial DNA loading dye. The homemade stuff from the wiki does not have this problem.

4. Protein purification

• When using spin concentrators, before adding protein, put a little water in and centrifuge, just to get the membrane wet. Otherwise your protein will stick to the dry surface and not come off.
• After spin concentrating protein, vortex the tube to get the protein off the surface.
• If you’re cleaving a solubility domain after purifying (like MBP or SUMO), take a sample of the protein before starting the reaction. The whole point is to know if it worked.
• When growing up a pellet, grow twice as much as you intend to use, so if the purification fails for some stupid reason you can try again immediately without having to start from scratch.
• Do not start a purification if you can’t start early in the morning. They take forever, no matter what.
• Keep all fractions from a column (either FPLC or gravity) until you run a gel and can know for certain which ones contain your protein.
• Run a sample of every single peak and shoulder from the FPLC, even if it doesn’t make any sense for your protein to be in any of them.
• When many fractions are run on an SDS-PAGE gel and most of them contain or might contain nothing, and one lane in the middle has something of interest, it’s impossible to tell which lane it’s actually in. One possible solution: add some ladders to every 3rd or 4th lane just to make identification easier.

5. PCR

• The surface of the liquid in the PCR tube must be lower than the metal heating block, or your reaction won’t heat properly.

6. pH Strips

• Detergent can lower the apparent pH of a buffer on pH strips by SEVERAL UNITS.

7. FPLC

• When you run a program on the Akta Purifier 10, if you pause and give it different instructions, that doesn’t stop the program. It will just do whatever you told it to do until the time for the next step of the program, and it will then resume doing whatever it wants. Always press “End”.
• Programs will pick up where they left off if you don’t end them. Again, always press “End”, so the next time you run it, it won’t start in the middle of the program.
• Claim a whole day if you need to do an overnight step.
• Run pump washes with elution buffer before you load your protein. When you do pump washes, small amounts of buffer will be sent into the column regardless of whether it’s programmed to do so.
• If you pause the Akta Purifier 10 and run a pump wash, it will continue with whatever it was doing before you paused it as soon as the pump wash is done. This can put some unwanted buffer into a column, so end it if possible or switch to the next step before the pump wash.

8. Worldview

• Just simply did not believe my data when it turns out it was capturing a real phenomenon