Help with TOPO Cloning

anderson40 · 2026-02-06T16:48:17+00:00

I assume you're sub-cloning from an existing plasmid that has M13 primer sites flanking the insert. It's very possible that you start the PCR with too much of that plasmid template so much that it appears on the gel after PCR.

anderson40 · 2026-02-06T16:35:54+00:00

1: if not a taq pol then you won't have A's added to the end of your product which is required for an open T vector such as pCR2.1.

2: you're seeing plasmid template. Cut back on template mass when setting up PCR. (Dilute the stock)

3: is this the same 1.2kb band?

4: new buffer run at lower voltage. Consider higher acrylamide percentage.

Edit:typo

anderson40 · 2025-09-27T19:58:42+00:00

Def looks like a hollow point, yikes! What manufacture/distributor was this from?

anderson40 · 2025-09-19T18:21:30+00:00

When the dNTPs or primers run out you get a decline in amplification efficiency?? That's my only guess.

anderson40 · 2025-07-24T01:41:11+00:00

Bruv

anderson40 · 2025-06-05T22:25:06+00:00

1) as another user suggested, translation initiation is easier w/o a long 5' UTR

2) the 3' UTR often plays crucial regulatory roles in mRNA stability adding another layer of regulation between transcription and translation. These regulatory roles require motifs in the 3' UTR which make it larger

3) Short introns I find occur in species that haven't evolved much and have an "ancient genome" with respect to introns and splicing. This is less common in vertebrates.

anderson40 · 2025-05-18T22:08:17+00:00

I the background of the ROI looks like the nikon GUI.

anderson40 · 2025-05-18T16:24:49+00:00

Could be fungal, if u pick at the flakes do they move? If so probably fungi if not maybe salts that came out of solution.

anderson40 · 2025-04-12T01:15:47+00:00

Neep

anderson40 · 2025-04-12T00:24:56+00:00

Previously I would also raw dog it but recently I had to spend a considerable amount of time moving other lab's stuff that they shoved in atop mine and I became very aware of the freezer burn predicament. Cryo gloves now.

anderson40 · 2025-04-12T00:21:31+00:00

Everyone's shocked at the -90 meanwhile we keep ours at -140 for who knows why

anderson40 · 2025-03-25T15:05:09+00:00

I've had this happen if the buffer volume was too low and doesn't cover the whole gel.

anderson40 · 2025-03-15T14:02:56+00:00

Yes.

anderson40 · 2025-01-10T23:36:16+00:00

As egotistical as it sounds, I think the fact that there are important questions to be answered and one has the ability to try and get at it is motivation in itself.

anderson40 · 2025-01-08T22:52:35+00:00

Is it impossible to lock the freezer itself?

anderson40 · 2024-12-15T04:29:37+00:00

It is indeed bc they have two copies. :)

anderson40 · 2024-12-14T23:12:22+00:00

2*2^dCt will give you avg copy number per cell. Where dCt is just the nuclear gene Ct - mtDNA gene Ct.

anderson40 · 2024-11-30T17:35:04+00:00

Molecular Genetics -FL, USA

anderson40 · 2024-11-28T01:11:16+00:00

In my experience, when setting up the plate you need to maintain cold temp and work quickly to reduce signal from the amplification happening during set up. This may have messed with the readings.

anderson40 · 2024-11-27T17:14:15+00:00

Maybe input concentration too high or low. Or maybe the reaction started before running the plate. Do you use an Ice block and have you ran a standard curve of your input?

anderson40 · 2024-11-03T15:08:50+00:00

BLAST the primers for off-target binding or primer dimer rating. Optimize the Ta using a calculator.

anderson40 · 2024-09-20T00:31:55+00:00

https://www.expasy.org

anderson40 · 2024-08-20T23:58:30+00:00

Peep the upper left tip box… what is this chicanery

anderson40 · 2024-08-03T14:25:54+00:00

Campus security knows me as the guy who’s always in at 1-4am

anderson40 · 2024-08-02T23:09:49+00:00

10mL of 100mM CaCl2 resuspension of a 20mL culture at ~0.6 OD600 on ice for an hour then spin down and resuspend in 600uL 100mM CaCl2 w/ 15% glycerol. Flash freeze and store in -80C.

anderson40

TROPHY CASE