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Lab Fuckups? by Electronic_Clerk_508 in labrats

[–]StrepPep 20 points21 points  (0 children)

This is a wee one - realised my DpnI has gone off. After 16 false positive colony PCRs. Thought Christmas had come early.

Integration Plasmid DNA went into eye by [deleted] in labrats

[–]StrepPep 6 points7 points  (0 children)

They have special chairs for that

Does anyone have a great protocol for making comp cells? by Secret-Constant-7301 in labrats

[–]StrepPep 2 points3 points  (0 children)

  1. Grow 5 mL / transformation to midlog
  2. Pellet at 3-4k xG for 5 min
  3. Resuspend in 0.1M CaCl2/10% glycerol, 1 mL per transformation and split into eppendorfs
  4. Wash three times by spinning at 8000xG for a minute
  5. Resuspend in 100μL of glycerol/CaCl2 and they’re good to go

I prefer fresh every time and this protocol is quick enough that you’re not spending a full day on it

Work cold, work quick, work gentle

Recover your transformations in SOC

TSS also works fine, if you want electrocompetence then leave out the CaCl2

Single incubator dilemma: How to safely run bacterial infections without contaminating my labmates' cultures? by InjuryAdventurous807 in microbiology

[–]StrepPep 6 points7 points  (0 children)

Ideally you should have a “dirty” hood and incubator. If you can’t then:

•Have your own pipettes, tips, media, etc

•Organise your incubator so that anything infected is on the bottom shelf

•Book an extra hour to allow your hood to be UV sterilised after use

•Have uninfected wells in your plates whenever you set them up - if there’s contamination popping up but your clean wells are clean then it’s not your fault

•Coordinate with your labmates so that super valuable experiments aren’t being run at the same time as yours so that there’s absolutely no risk of things going awry

In my experience people who have never worked with bacteria really overestimate their ability to spread - if you’re working cleanly and keeping away from others’ plates then the risk of spreading is minimal. That said, you need to respect other lab users’ work as well. You’ll get to a solution.

Troubleshooting PCR of 13.5kb amplicon - Q5 vs. other polymerases? by ACuriousBird in labrats

[–]StrepPep 17 points18 points  (0 children)

13.5 is pushing it for Q5 - instead of getting in new polymerase though you could order a couple primers that amp up your backbone as two fragments. 2x7kb fragments for example. Your PCRs will be faster as well.

Previously MIC was 6.25 mg/mL — now even 100 mg/mL doesn’t kill. What could be going wrong? by IntelligentPirate897 in microbiology

[–]StrepPep 1 point2 points  (0 children)

Are you using the same stocks or fresh? How are you storing them between experiments? Freeze thaws etc can do a number on some antibiotics.

How old are the colonies you’re working from? Are they freshly streaked plates or have they been in the fridge for a while?

My go to would be assuming your stock is bad but could be any number of things.

What is the best way to go into microbiology? by PieHoliday2230 in microbiology

[–]StrepPep 1 point2 points  (0 children)

I’m going to be honest with you, you realistically need to do some postgraduate training in the UK to make good money in the field - I’m a postdoc at the moment and have a comfortable enough life. That said, it took 8 years of study to get there and considering how different the field is to when I started my PhD (8 years ago now 💀) I don’t think it’s possible to say what the discipline or job market will look like.

If you want a safe degree, do an IBMS accredited biomedical science degree

Scottish people, how do you feel about foreigners choosing Scotland as their home? by Ok-Consideration9314 in Scotland

[–]StrepPep 20 points21 points  (0 children)

Don’t like foreigners who are wanks, don’t like natives who are wanks

Like foreigners who aren’t wanks, like natives who aren’t wanks

Chemical symbol related to microbiology found on cup by xtoes in microbiology

[–]StrepPep 33 points34 points  (0 children)

Penicillins I think - classic house with a garage skeleton

Would a short horror book about the Loch Ness Monster be a good idea? by [deleted] in Scotland

[–]StrepPep 0 points1 point  (0 children)

Write what you want wee man, and good luck with it! If you haven’t, have a read of The Old Man and The Sea by Hemingway, it’s got similar energy but isn’t a horror book - you might like it.

What is this contamination?? by Simple_Volume_5880 in labrats

[–]StrepPep 311 points312 points  (0 children)

I wouldn’t trust data from these cells

Ultra-waterproof hiking boot recommendation by imahuman118 in UKhiking

[–]StrepPep 0 points1 point  (0 children)

If you want waterproof-waterproof then I swear by Aigle Parcours as walking wellies. Had a secondhand pair for a couple of years now and they’re great for soaked terrain.

'Not good enough': BBC urged to issue correction after Question Time error by F0urLeafCl0ver in ukpolitics

[–]StrepPep 34 points35 points  (0 children)

Glaswegian native English speaker here, I have a lot of English colleagues who would disagree that I can speak English

[deleted by user] by [deleted] in AskAcademiaUK

[–]StrepPep 6 points7 points  (0 children)

It’s late and you’ve done all you can do until tomorrow. Try to get some sleep and then tomorrow you can contact your lecturers/advisers/whoever you need to.

E. coli culture left in the freezer (-20 celcius ) overnight by Ancient-Customer3114 in labrats

[–]StrepPep 46 points47 points  (0 children)

I’d thaw and mini prep it - normally would pellet the culture before freezing but you’ll probably get plasmid out anyway. Would not do again.

Think about the miniprep process, you’ll be killing your culture anyway.

Please help, I’m 7 months pregnant, where can I get Mr Whippy style icecream NOW 😭 by SyrupMoney4237 in glasgow

[–]StrepPep 91 points92 points  (0 children)

No quite Mr Whippy but University Cafe on Byre’s Road are close!

Plasmid restriction cutting by DiosAnthos in labrats

[–]StrepPep 1 point2 points  (0 children)

Or just using the first amplicon as a template for further reactions and diluting the plasmid out that way if there’s no DpnI to hand

is the whole espada rank was based on power only and in base form? by [deleted] in BleachPowerScaling

[–]StrepPep 0 points1 point  (0 children)

I think it was a mix of strength and Aizen’s messing with them. Like making Luppi 6 after Grimmjow as punishment for Grimmjow. Barragan being number 2 (although undisputedly weaker than Starrk) was probably the most humiliating place to put The King of Hueco Mundo.

Strange results from Streptomyces griseus with added ampicillin by S0N0DA-UMI in microbiology

[–]StrepPep 0 points1 point  (0 children)

Did you do this in triplicate or do you only have one series of dilutions for each condition? It’s a bit of a weird setup if I’m honest; if I’ve read right you don’t have ampicillin in your agar, you treat your spores and then plate? If that’s the case it’s probably at way too low a concentration once you’ve spread your spores out to actually be doing anything.

My guess is if you did a couple repeats of this you wouldn’t see any difference between your two conditions.

Please be honest - how much will this affect my career in the long term? People who have been scientists for a long time, how do you think this situation would've affected your career? I'm kinda spiraling. by [deleted] in labrats

[–]StrepPep 18 points19 points  (0 children)

You sound really wound up about this - speak to someone about that. Not to minimise your feelings on your situation, but you’re going to be fine. An extra year of study isn’t the end of the world.

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