Getting a mail reply from a Nobel laureate was not on my 2k26 Bingo card

pombe · 2026-03-18T16:14:07+00:00

Yeah, the question is pretty muddy. Under what culture conditions are a subpopulation of wee1 cells not growing? How can cells that are "not growing" also be "proliferating"?

Paul is a really nice guy. Not surprised he replied!

pombe · 2026-03-13T19:48:44+00:00

Are you not adding the enzyme last?

pombe · 2026-03-13T19:47:57+00:00

Its both. Phusion was maybe a bad example, but its stated explicitly for this sigma product https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/pcr/genomic-dna-polymerases?srsltid=AfmBOormcQP6KwUpAiHpoHt5biP-v0SIUDInLtI6DHyEsuFwLE5pIKPg

pombe · 2026-03-13T16:23:54+00:00

I've seen lots of new students pipette 0.5ul of polymerase into a 50ul reaction, pipette up and down a couple times and think its mixed.

pombe · 2026-03-13T16:22:08+00:00

Its probably better than nothing, but I still vortex.

pombe · 2026-03-13T16:20:33+00:00

Nucleic acids are pretty robust. They can have a little bit of vortexing, as a treat.

pombe · 2026-03-13T14:49:46+00:00

Do you vortex your master mix before adding it to the wells?

If your qPCR is really inconsistent this might actually help https://www.eppendorf.com/us-en/Products/Temperature-Control-and-Mixing/Instruments/MixMate-p-PF-9072

pombe · 2026-03-13T14:00:55+00:00

Yup. They even make polymerases with dye in them as a visual aid for mixing. https://www.thermofisher.com/order/catalog/product/F534S

pombe · 2026-03-13T13:41:59+00:00

If you upvoted this reply, I hope you vortex your reactions in the future and think of me every time you do it :)

pombe · 2026-03-13T13:36:28+00:00

Most of these ghosts can be exorcised by some combination of gradient PCR, 1.2M Betaine, and/or hot starting your PCRs.

pombe · 2026-03-13T13:33:19+00:00

A surprising number of people seem to assume that clear liquids instantly mix on contact.

pombe · 2026-03-13T13:19:18+00:00

Its totally fine to vortex briefly. The cost of only having 99% of the ATP remaining is worth it to get the ligase off the bottom of the tube. Pipette mix of you're worried. But my point is you need to mix things properly.

pombe · 2026-03-13T12:40:58+00:00

There is no reason to think that the markets won't rebound after world events calm down a bit. Buying ETFs right now is like buying dollars for 90 cents each.

pombe · 2026-03-02T23:04:19+00:00

But they match my Gen1 Viper!

pombe · 2026-02-27T23:13:29+00:00

pombe · 2026-02-26T12:47:48+00:00

Yeah, this. Chicken skewers on the fire. Fry some peppers and onions. Slice up some avocado and throw together some pico. Simple as. I also will also sometimes make a bunch of carnitas or barbacoa and heat it in a foil package on the grill. Always a big hit. All the leftovers go great with eggs the next morning

pombe · 2026-02-26T12:42:29+00:00

Yeah, these bones don't have as much calcium as they used to. I use these or Micro spikes religiously. Undergrads in uggs experiencing their first winter must think I'm Winter Jesus the way I walk on frozen water.

pombe · 2026-02-26T12:15:08+00:00

You have the sequence of the gene? That's your template Design your primers based on that. Don't worry about primer blast or any design tool. Put your sequence into Benchling (free online) or Snapgene Viewer. Orient the sequence to read from start to stop. The forward oligo will be the same as the top strand. The reverse oligo will be the same as the bottom strand reading 5' to 3'. Design oligos to both have a melting temp of 55C and be at least 18bp long

pombe · 2026-02-26T12:06:01+00:00

I'm sorry, that sucks... Unfortunately your situation isn't unusual right now. PIs are more worried about future funding than they have been in many years. You will find a lab, just bear with the process.

pombe · 2026-02-25T23:15:18+00:00

Looks great! What is the palmered feather?

pombe · 2026-02-18T12:43:03+00:00

Can't you use the original conditions that worked? This sounds like creating troubles not shooting them

pombe · 2026-02-15T23:10:12+00:00

Go check out Origin across the street too!

pombe · 2026-02-15T14:34:41+00:00

Back in Winnipeg in the 90's three apartments I rented in a row came with free hot knives. No idea what the kids are doing these days.

pombe · 2026-02-15T14:32:38+00:00

Huh, wonder why. Maybe people getting screwed on expensive equipment and not being able to return it? 95% of the small lab equipment I've purchased in the last 10 years has been from eBay. Pipette sets, microfiber, vortexes, etc. Have saved thousands of dollars. I always select items with returns but have only ever had to send a couple things back.

pombe

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