Janet B. Matsen:Best Lab Practices
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Efficiency Versus Thoroughness
I have really come to appreciate the trade-off between thoroughness and efficiency/throughput. If you are overly thorough and careful, things will get done too slowly. If you are overly throughput oriented, errors will occur and you will waste too much time figuring out what went wrong. I am always looking to tune my sweet spot in all of my lab practices, and always looking for tips from others.
- Page numbers are usually more useful than dates. I put page numbers (and usually dates) on all tubes I put in the freezer.
- Keep a digital list of every primer you order that you can query and reference. I number each primer tube and have them arranged chronologically in my freezer.
- I prefer to use a sketchbook rather than a lab notebook. You can buy them cheaply at art stores, they have bigger pages, and most importantly, they don't have distracting lines. This allows me to flip through pages and look for shapes (eg a table of DNA concentrations or a particular gel) more quickly.
Committing to the freezer
- When you sequence a strain and find you created what you intended, it is time to "commit" it to the freezer. It is possible/likely that you had several incorrect constructs sequenced along the way. In general, how best can you keep organized so that you don't ever store the wrong strain, keep an incorrect plasmid, etc? I am really excited about the flow I developed for myslef:
- Take a single colony that you will screen and inoculate two cultures with the same stick. One will be for a DMSO/glycerol stock at -80oC and one will be for miniprepping.
- The next day when your cultures have grown up, miniprep one culture and archive the other in a DMSO stock at -80oC with the description, date, and page number on both tubes. These should both go in "pending" boxes where only un-verified constructs live; the DNA will be stored at -20oC and the cells at -80oC.
- When you get your sequencing results back, you move each tube to a new box. If the sequence confirms your construct, move each to a special "verified" box at its respective temperature. Give each "commit" a unique number and document it in a google spreadsheet. Cultures that are not perfect get moved to "trash" boxes that you can hold on long enough to be *sure* it is trash.
- I'll make a diagram of this system.
"Did I do that?" (Bench Work)
- Often we do things with many tubes in parallel, such as screening colonies. One of the worst feelings is having a moment of doubt whether you pipettted into the right well in a strip of PCR tubes or whether you added a particular reagent to a master mix tube. It can happen because tubes in the middle of a row are hard to distinguish, or because a though or conversation distracts you. Tricks I have enjoyed to prevent this:
- having dogmas about the order you do things in such as "always add VF2 before VR," "always handle the tubes left to right," and "put pen dots next to reagents/steps you have added/done in a list of ingredients/steps."
- moving my eppendorf tubes from one location in the holder to another once you have taken/added material
- makes sure you don't forget one or do one twice
- drawing on PCR tubes so the wells are grouped in sets of 2-4, or by numbers relevant to how I want to order my samples.
- This gives me a point of reference when I pipette precious samples around. For example, if I am screening 7 colonies from project A, 4 from project B, 2 from C, and 8 from D, I will use a sharpie to mark the plastic after the 7th, 11th, & 13th tubes. Then as I put my samples into the PCR tubes I will know I am getting one sample in per well and have not skipped a well or put two samples in one well.
- Use a multi-pipette if you have one available and you are working with many tubes (such as PCR tubes)
- This will greatly reduce errors caused by injecting things in the wrong tube.
- write out the recipe for a master mix, and put a dot next to ingredients you have added... every time.
- I used to do this on occasion, but I recently decided to commit it to my "always" list. This is the quickest way to diagnose a failed PCR. Just recently I was *sure* I added everything but go no bands. Sure enough, I looked at my notebook and saw there was no dot next to "template." Couple this trick with "always check that each item has a dot before you use it" and you are golden!
- If you are doing something at all complicated, make a cad design of it before you start your DNA work. This will make you sure you have your design right, allow you to check your primers properly, and be available for consultation/sharing at a later date.
- Science should be fun! Treat everyone well and keep your workplace a pleasant place for everyone to come.