I thought I should write down tips that I have discovered throughout years of doing science.
- When performing dilution (e.g. from 500 mM imidazole to 15 mM, for 50 ml final size), sometimes it is a good idea to add small volume (1.5 ml of 500 mM) into an empty conical tube, then add the rest of the buffer (48.5 ml of PBS). Adding large volume of liquid into small volume of liquid kinda mixing things, so does not require an elaborate mixing afterwards, i.e. swirling the tube.
- When making buffer, it is always a good idea to make 10-20X stock instead of making 2X stock. Sometimes, the amount of powdered reagent to make 2X stock is just way too small.
- Pre-wet micropipetter tips! I usually go for 5-7 times when performing serial dilutions for serum/plasma samples. Sometimes, things (especially stickier ones like antibodies) get stuck in the pipette tips, hence pre-weating sorta kinda compensates that.
- Sometimes, pre-wetting up & down forces the fluid to back-flow into the pipette tip. There are 2 potential countermeasures for this: (1) push down and pipette up gently, but ain’t nobody got time for that, so; (2) pipette up gently to avoid the liquid from jumping up, and when about to push down, raise the pipette tip just above the surface of the liquid. That way, it would reduce the chance for forced back-flow.
- When dispensing serially-diluted solutions, start with lowest concentration (i.e. highest dilution) first, then go up serially. This would allow you to use the same tips, therefore reducing plastic waste.
- When preparing reagents in 25-50 ml reservoir, plan for 500 µl buffer or more. It is especially important when preparing the reagent in 50 ml conical tube to use serological pipette to transfer from conical tube to the reservoir to get around the dead volume due to liquid sticking around in the tube. Alternatively, mix reagent within the reservoir itself. For multi-channel reservoir, usually 200 µl buffer is fine.
- To warm up growth media, I usually tend not to submerge the whole 500 ml bottle in water bath. It feels a little… unhygienic. What I would do is start adding the growth medium into the intended culture container (e.g. 250 ml flask), then let it sit in the 37 °C incubator for a while (30-60 min). Or, add the desired amount of media in 50 ml conical tube, then place in water bath, then discard after use. It is a good idea to avoid placing the main media container in water bath. Maybe I am just paranoid.
- When counting cells with Trypan blue, n=50 translates into 1 million cells/ml; n=100 (translates into 2 million cells/ml) is on the hemacytometer is my upper convenient limit, so plan the dilution accordingly.
- Phusion polymerase (NEB) tends to work the best for me, and has higher efficiency than Q5 (NEB). I tested this side-by-side, and got thicc band with Phusion.
- It is always a good idea to run multiple conditions (e.g. gradient of 6-8) when testing a potentially problematic primer, than having to run over and over again several times.
- When making master mix from scratch, I usually make separate mix for reaction buffer, polymerase, dNTPS, and/or DMSO (final of 3%). Primers (fwd and rev) will be in separate tube.
- When pipetting the primers and master mix, I usually pipette them to the wall of the tube. The droplet would stick to the wall, hence serving as a nice visual confirmation. Also, this allows me to not change tips when dispensing into multiple tubes, reducing plastics.
- For high-throughput loading of DNA (mixed with loading dye) into casted agarose gel, instead of using Eppendorf tubes to mix DNA and dye, use parafilm strip instead. Take out 2 µl of DNA and drop it on a parafilm strip, then mix with 0.7 µl of load dye (if dropped carefully, no need to change the tips between samples). Then, use 1.5-1.7 µl of the DNA:dye mix to load into casted agarose gel (small well).
Protein expression and purification
- When doing small scale purification (e.g. 30-50 ml supernatant of secreted protein), just use syringe (20 ml or bigger) with syringe filter (0.45 µm pore size is fine) to filter cell debris. No need to use vacuum filter bottle.
- When running gravity column purification (e.g. with BioRad Glass Econo-Column) and running beads/resins in it, best to have the beads settled down in mixing container (e.g. 50 ml conical tube) and start by transferring the liquid supernatant. Column would run faster this way. Once the beads accumulate and settle down in the column, the flow speed would be slower, thus would take more time.
- Please pipette carefully (e.g. place tip on the wall). If you make bubbles or air buildup, the column would run slower, and would require some violent mechanical perturbation (lol) to dislodge it.