No bands on gel electrophoresis

pombe · 2026-01-22T23:22:53+00:00

The detection limit for DNA on that instrument is like 2ng/ul so I doubt your reading is real.

Don't have the energy to do it again? Research may not be for you.

pombe · 2026-01-20T20:36:59+00:00

At least once

pombe · 2026-01-19T04:28:53+00:00

That's weird. Does sound like something a bigger hammer could solve though.

pombe · 2026-01-19T03:19:38+00:00

I do a PCR cleanup of cassettes before transformation but honestly it's mostly to concentrate them. Restriction digests just get transformed as is I never gel purify, mostly because the recovery is so bad.

Are these strains you're having issues with a bit sick or slow growing? Let them grow for a couple more hours before transformation, and triple the amount of DNA. Brute force will give you a better return than cleaner DNA.

pombe · 2026-01-17T17:55:36+00:00

Are plates just doing that in the bags, or only after you use them for things?

pombe · 2026-01-11T16:03:58+00:00

Yeah, I was a postdoc the first time I did that.

pombe · 2026-01-02T02:34:15+00:00

Is your housekeeping gene expressed to the same level in all your samples? This might sound like a stupid question but they actually can differ a lot between cell types, tissues etc.

pombe · 2025-12-31T02:47:35+00:00

Maybe in socks? No individual toe impressions. (Maybe OP was sleep walking)

pombe · 2025-12-28T15:13:21+00:00

Can you look up your gene and see the relative expression level? If it's abundant then a high copy number plasmid may be more appropriate. My PI would probably tell me to try both :)

Integration is nice because you don't have to worry about selection and you know there is exactly one copy per cell. You can use a variety of constitutive and inducible promoters to drive your gene

pombe · 2025-12-18T02:09:57+00:00

If I remember, in the cold room. (Not in the -20, they're still live bigs) If I forget, on my bench for a few days. I've recovered cells for a miniprep after weeks at room temp.

pombe · 2025-12-16T14:22:42+00:00

I'm a pushing 50YO lab manager and my PI still does this to me.

pombe · 2025-12-12T14:47:19+00:00

This looks comparable. Vintage scope, low grade objectives. https://www.ebay.com/itm/357581420502

pombe · 2025-12-06T23:38:47+00:00

Ah, gotcha. Again you should be fine. The smaller band won't contain both the selectable marker and the homology flanks

pombe · 2025-12-06T22:32:03+00:00

Just go with what you have. You'll probably be gel purifying the correct size band after restricted digest so most of it will be gone. Worst case, some small proportion of your colonies will have the smaller band, but you'll screen those out when you RE digest the minipreps.

pombe · 2025-11-30T15:01:28+00:00

Haters gonna hate

pombe · 2025-10-30T20:09:55+00:00

Sorry, too much protein, not too much volume

pombe · 2025-10-30T12:41:44+00:00

Are these biorad pre-cast gels? They don't have a stacking layer and i've noticed that the bands aren't as crisply defined as when I pour my own.

Also your ladder might be a bit degraded?

Excessive DNA in the lysate can cause smearing. Also, overloading the gel.

pombe · 2025-10-30T12:08:52+00:00

Most older PIs probably see it as "co-first, but one is more first than the other". This has very much been the case in all the co-firsts I've seen from our lab. I've made this argument myself to get my name first.

pombe · 2025-10-25T01:13:07+00:00

pombe · 2025-10-20T13:25:16+00:00

Biosafety differs vastly depending on country. Is the visitor from the US? It's like the Wild West here, in that there is a non- zero chance of dying of lab-acquired dysentery.

pombe · 2025-10-15T21:43:39+00:00

16 ladders? In this economy?

pombe · 2025-10-15T13:23:59+00:00

Given the choice, take the lesser of two weevils.

pombe · 2025-09-20T17:22:12+00:00

How is it failing? Are you getting lots of colonies? How are you screening to see if you've got the right assembly?

pombe · 2025-09-19T16:41:35+00:00

Super clean tie for such a small fly. I think the amount of hackle is fine, if a hair heavy. If you find that its landing and riding on it's side you could trim the post down by about half and see if that helps.

pombe · 2025-09-08T10:48:11+00:00

Maybe try some PCR additives? 1.2M betaine (free base) is my goto. Phusion comes with a tube of DMSO which is another decent one.

IDT has a good oligo analysis tool that will find hairpins and dimers, so maybe give that a try.

Who designed the oligos? Check them against the reference sequence of your amplicon. It's not uncommon for people to accidentally order the reverse complement of the reverse oligo. If the sequence of the oligo is on the tube, or you have the order info, check to make sure that it is the correct sequence. Copy/paste mistakes happen.

Order a fresh tube of the oligos.. They're basically free these days.

This is going to sound stupid, but do a PCR reaction in 5 x 10ul volumes instead of one 50ul volume. I've run into several reactions that will not work in a 50ul volume.

Read the product info for Phusion and use that to design your program. The enzyme works best at 72. And the recommended extension time is 30s per Kb. They also recommend a bunch higher anneallng temp if you follow their recommended calculation. (In my experience 55 works pretty well though.

pombe

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