But this will be 90% of your job, so pay attention. 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). Agar can be left in the water bath for hours.Īnother “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.Īpparently, some primers degrade if left at room temp. Use your sterile technique, be quick, and it’ll be fine.ġ2. Turn it off and gently clean out all those salts and powders. If it’s taking too long for the lab scales to tare, it just might need a good scrubbing. 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? If you have your own box with your own aliquot of that enzyme, your experiments are unaffected. 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. Need more time? Leave them on ice longer than 30 minutes.
TOUCHDOWN PCR MICROBIOME FULL
Running short on time? They just might be fine after 45 minutes of recovery, rather than a full hour. Too few colonies? Spin down the cells (gently), resuspend them in a lower volume, and plate them all. Too many colonies? Reduce the 30 minute incubation on ice to 15. Chemical transformation protocols are gloriously flexible. 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. 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. The plasmids are so clean and friendly! And it’s easy to share sequences, alignments, even entire DNA libraries with colleagues. Benchling is so much more aesthetically pleasing, more intuitive for exploring DNA. Using them only resulted in messing up my primer orders. This is another good reason to use TB – hit those high ODs in shorter periods of time.Įnough with those unreadable DNA “visualization” softwares. Which reminds me, don’t let that culture grow too long – you risk the cells picking up some annoying mutation. TB is a richer media, and cells grow to higher ODs and faster in it, given the same incubation time. There is no reason to use LB unless you want to elute low amounts of DNA. 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. Oh, add the solution and incubate at 5 minutes, you say? Ha. Melt agarose gels longer than the protocol tells you. And chances are, every time you mix a new batch, the cells will be pretty sensitive to even slight changes in its concentration.Ĥ. If adding to agar, make sure it is cooled to 55C ( and no warmer) before adding. Dissolve the flaky white salts that precipitated out of solution with a nice long vortex.
But if you do, melt it in your hand, or in the 30C ( never 37C), or just on your bench for a while. Ok, not all of you will have to work with this one. No shame in scrambling to find old plates in the fridge to see if your late night transformation worked. 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. 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. Primers bind more specifically at higher melting temperatures closest to their own magic number, but they bind more easily at lower temperatures. – 65C 15 seconds, subtract 0.5C each cycleĪnd then anneal for 10 minutes, hold at 10C. It requires a little thermocycler hack on the annealing step: But if even one is new, it might make your life a lot easier.Īre 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? Some of these may seem ridiculously obvious.
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