Some of these may seem ridiculously obvious. But if even one is new, it might make your life a lot easier.
1. Touchdown PCR.
Are you doing a massive PCR reaction set, with various primers that need all sorts of different melting temperatures? Are you doing a PCR with primers you never bothered to check the melting temperature for? Is your PCR reaction just not working in spite of everything you have tried to troubleshoot?
Look no further than a touchdown PCR. It requires a little thermocycler hack on the annealing step:
98C 2 minutes
– 98C 15 seconds
– 65C 15 seconds, subtract 0.5C each cycle
– 72C for X amount of time
– 98C 15 seconds
– 55C 15 seconds
– 72C for X amount of time
And then anneal for 10 minutes, hold at 10C.
The trick is the first annealing step. Primers bind more specifically at higher melting temperatures closest to their own magic number, but they bind more easily at lower temperatures. So starting with higher temperatures allows the proliferation of a few correct template strands in the PCR reaction, and then at lower temperatures the primers bind more easily but to the correct templates that were created earlier. Works like magic every time.
2. Use those month-old plates.
Some guy in the 70’s tested how long antibiotics remain effective while stored in agar plates in the fridge, and turns out, they’re pretty darn stable. No shame in scrambling to find old plates in the fridge to see if your late night transformation worked.
3. Spectinomycin is a brat.
Ok, not all of you will have to work with this one. But if you do, melt it in your hand, or in the 30C (never 37C), or just on your bench for a while. Dissolve the flaky white salts that precipitated out of solution with a nice long vortex. If adding to agar, make sure it is cooled to 55C (and no warmer) before adding. And chances are, every time you mix a new batch, the cells will be pretty sensitive to even slight changes in its concentration.
4. Melt agarose gels longer than the protocol tells you.
Oh, add the solution and incubate at 5 minutes, you say? Ha. The protocol lies. Leaving it in longer – thirty minutes, say, or even up to an hour – will help you avoid the disastrously low DNA concentrations that gel purification protocols are famous for.
5. Use TB for miniprep cultures.
There is no reason to use LB unless you want to elute low amounts of DNA. TB is a richer media, and cells grow to higher ODs and faster in it, given the same incubation time. Which reminds me, don’t let that culture grow too long – you risk the cells picking up some annoying mutation. This is another good reason to use TB – hit those high ODs in shorter periods of time.
6. Benchling is free in academia.
Enough with those unreadable DNA “visualization” softwares. Using them only resulted in messing up my primer orders. Benchling is so much more aesthetically pleasing, more intuitive for exploring DNA. The plasmids are so clean and friendly! And it’s easy to share sequences, alignments, even entire DNA libraries with colleagues. And it’s free, if you’re in academia.
7. Label all your samples.
If you’re like me and somehow acquired sixteen different minipreps of that one plasmid you use all the time, be a champ and label each one uniquely. If one yields weird results (maybe it was left on the bench too long, or got contaminated, or something), you’ll know it was A1 that did this to you and not, say, B3 or C4.
8. Chemical transformation protocols are gloriously flexible.
Too many colonies? Reduce the 30 minute incubation on ice to 15. Too few colonies? Spin down the cells (gently), resuspend them in a lower volume, and plate them all. Running short on time? They just might be fine after 45 minutes of recovery, rather than a full hour. Need more time? Leave them on ice longer than 30 minutes.
9. Keep your own freezer box.
If Joey clumsily takes an enzyme out without ice, then returns it to the communal freezer, you’ll suffer next time you use it, without a clue as to why things failed. If you have your own box with your own aliquot of that enzyme, your experiments are unaffected. Contrariwise, maybe you’re the Joey in this hypothetical scenario, and in that case – why not keep those slip-ups to yourself, confined to your own little freezer box?
10. Scrub the scales.
If it’s taking too long for the lab scales to tare, it just might need a good scrubbing. Turn it off and gently clean out all those salts and powders.
11. Agar can be poured on a bench.
You don’t need a flame. Use your sterile technique, be quick, and it’ll be fine.
12. Agar can be left in the water bath for hours.
Another “running short on time?” trick – let it stay nice and liquidy after the autoclave cycle in the water bath at 55C, just until you finally catch a breath and can pour those plates.
13. Keep primers cool.
Apparently, some primers degrade if left at room temp. Not all of them do this. But some do. This may be sequence specific? (RIP my bad habits from an old job, where we all left boxes of primers on our benches for years). Keep them in the fridge instead.
14. Pipetting is an art form.
I know it looks brainlessly easy. But this will be 90% of your job, so pay attention.
15. Plan each day.
Start each day – each week, if possible – with a plan for all your experiments. Bonus points for including fluidity of connections between each one, or how one changes depending on the results of another. Build trees, forests even – not piles of sticks.