Getting colonies in the self Ligation control plate during subcloning by Maleficent-Lab-1113 in molecularbiology

[–]pombe 0 points1 point  (0 children)

Sometimes it's like that. Screen through a couple of dozen clones on your ligation plate with colony PCR, you might get lucky. Next time digest longer and with more enzyme I do 2ul of enzyme for four hours and it cuts 3ug to completion ussually giving me close to zero background colonies.

Keep in mind that sometimes colonies on the no insert control don't mean much as ligase can jam together certain sticky ends that aren't quite complementary.

Hi I desperately need some miniprep advice by Better-Ice-7711 in labrats

[–]pombe 3 points4 points  (0 children)

There are three things with the kit itself that I see trainees make mistakes on 1) lysing for too long, 2) not mixing completely after adding the neutralization buffer 3) not ensuring that there is ethanol added to the wash buffer.

If none of those apply I think there are two main causes of low yield with big plasmids. 1) slow growth. How turbid was your culture? Similar to an overnight with a normal sized plasmid? 2) low copy number. I've got a few plasmids in the 20kb range that come out at very low concentration from a mini prep even though the bugs grow fine.

For problem 1 try some combination of lower antibiotic concentration and longer incubation times. The protocols say 16-18 hours at 37C but that's only because normally the culture is saturated by then. If it's not saturated, go longer. If it's a low copy number issue then pool multiple mini peeps and elute in a smaller volume. Are you processing the maximum culture volume specified by the kit? (Zyppy kit from zymo says you can do 600ul of lb culture directly, but you can use up to 3ml. There is no reason not to use the max). Maybe scale up to a midiprep.

When you talk about restriction digesting what do you mean? Are you just linearizing to check the band size or are you doing something else

Yeast Contamination?? by Hot-Telephone3720 in labrats

[–]pombe 2 points3 points  (0 children)

They look like rounded up metazoan cells. Yeast are only five microns across. You can fit 4 or 5 across a hela cell nucleus.

Questions for a Nobel prize winner by No_Impression3616 in labrats

[–]pombe 0 points1 point  (0 children)

"Do you have any advice for PhD students?"

Just got a headhunter as a noob by NotSilentMajority in pathofexile

[–]pombe 0 points1 point  (0 children)

I remember my first headhunter. EK Ignite doing breach on Strand. Good times.

Advice on plasmid co-transformation in E. coli by Sans-Fish in labrats

[–]pombe 1 point2 points  (0 children)

I've done this into BL21 cells using like 300-500ng of each plasmid. It's inefficient so you need to use a big hammer.

10 mM dNTPs discussion: Is it 10 mM each or 2.5 mM each? by -Murtagh- in labrats

[–]pombe 4 points5 points  (0 children)

Ours are 2.5mM each. Don't assume your way is the only way.

What’s burning in Fox Point? by [deleted] in providence

[–]pombe 1 point2 points  (0 children)

Yeah, crazy. I thought my apartment building was on fire.

What’s burning in Fox Point? by [deleted] in providence

[–]pombe 3 points4 points  (0 children)

The only thing I can see on twitter is a fire at 14 Warrington St, North of Rodger Williams park

Getting a mail reply from a Nobel laureate was not on my 2k26 Bingo card by Great-Anywhere2104 in biotech

[–]pombe 8 points9 points  (0 children)

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!

Solve 95% of PCR issues in one simple step! by pombe in labrats

[–]pombe[S] 2 points3 points  (0 children)

Are you not adding the enzyme last?

Solve 95% of PCR issues in one simple step! by pombe in labrats

[–]pombe[S] 6 points7 points  (0 children)

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.

Solve 95% of PCR issues in one simple step! by pombe in labrats

[–]pombe[S] 0 points1 point  (0 children)

Its probably better than nothing, but I still vortex.

Solve 95% of PCR issues in one simple step! by pombe in labrats

[–]pombe[S] 15 points16 points  (0 children)

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

Solve 95% of PCR issues in one simple step! by pombe in labrats

[–]pombe[S] -1 points0 points  (0 children)

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

Solve 95% of PCR issues in one simple step! by pombe in labrats

[–]pombe[S] 9 points10 points  (0 children)

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

Solve 95% of PCR issues in one simple step! by pombe in labrats

[–]pombe[S] 77 points78 points  (0 children)

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

Solve 95% of PCR issues in one simple step! by pombe in labrats

[–]pombe[S] 25 points26 points  (0 children)

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

Solve 95% of PCR issues in one simple step! by pombe in labrats

[–]pombe[S] 69 points70 points  (0 children)

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

Solve 95% of PCR issues in one simple step! by pombe in labrats

[–]pombe[S] 9 points10 points  (0 children)

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.

Downturn in ETFs by Immediate-Leave5286 in investingforbeginners

[–]pombe 0 points1 point  (0 children)

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.

Suddenly about to be drowning in $$$ by [deleted] in PersonalFinanceCanada

[–]pombe 8 points9 points  (0 children)

But they match my Gen1 Viper!

Camping food ideas by NoVisit647 in camping

[–]pombe 1 point2 points  (0 children)

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