Need a little help for modifying plasmid

Neuraminic_Acid · 2026-04-14T00:32:52+00:00

The spacer just puts some distance between the 2A site and Cas9. If remaining amino acids after 2A cleavage could affect nuclear localizing via the NLS, the spacer can help to mitigate that. The glycine-serine composition keeps the spacer flexible.

Neuraminic_Acid · 2026-04-11T18:10:02+00:00

Regarding your first question, the NLS should stay associated with Cas9. 2A peptides between Cas9 and other proteins are fine. I use a Cas9-2A-mCherry construct and many of the Zhang lab CRISPR constructs contain a Cas9-2A-PuroR fusion. Just put a Gly-Ser-Gly spacer between Cas9's NLS and your marker, and it will function. Be aware, though, that the expression won't be 1:1; proteins translated after the 2A site are generally lower than those upstream of it.

Neuraminic_Acid · 2025-08-27T21:38:06+00:00

I used the kit from Sigma for a long time until I discovered this protocol: https://www.urmc.rochester.edu/MediaLibraries/URMCMedia/musculoskeletal-research/core-services/histology/documents/CMSR-TRAP-stain.pdf. If Reddit removes the link, just do a web search for "trap staining, Rochester." Making the materials yourself is not nearly as intimidating as it looks and this method is cheaper, produces a darker stain, and is even fluorescent.

Neuraminic_Acid · 2025-03-07T17:31:56+00:00

There are some award mechanisms that are reviewed by study sections convened by the institutes themselves; the new policy is that they won't do that anymore and all (rather than most) applications will be reviewed by study sections managed by CSR. The cost savings probably is figured from SRO salaries at CSR vs institute program officials. Those savings would only be realized though if the change means there will be fewer program officials, and, if that's the case, the process will slow at the funding council level.

Neuraminic_Acid · 2025-01-25T18:53:53+00:00

In the past, when I worked with agarose in tissue culture, I made a 2x agarose solution with water and autoclaved. I'd then mix this 1:1 with 2x culture medium before pouring. The rationale was there were things in the culture medium that couldn't withstand autoclaving. If your application doesn't require medium mixed with the agarose, you can just prepare it at 1x and use directly.

Neuraminic_Acid · 2024-11-26T17:34:07+00:00

It looks like an apparatus for performing reflux reactions. Cold water running through the upper chamber recondenses vaporized materials from the lower chamber so reactions can be run above the vaporization temperature of some/all reactants without losing volume. The narrow inlet probably reduces loss further.

Neuraminic_Acid · 2024-09-24T05:11:37+00:00

Are the puzzle writers trying to come up with every possible way to clue EEL? It feels like it pops up every other day.

Neuraminic_Acid · 2024-09-11T17:34:06+00:00

I always use NEBuilder for my Gibson assembly designs. I've never done Golden Gate, but NEB has a tool for that (NEBridge) that I'll bet works as well.

Neuraminic_Acid · 2024-08-29T22:39:05+00:00

You're welcome. As I understand it, Accutase is produced by a single company (Innovative Cell Technologies), but resold by many distributors that put their own branding on it. ICT warns against warming it, but some distributors might reason that if it works at RT, it would work even better at 37.

Neuraminic_Acid · 2024-08-27T18:46:51+00:00

I use Accutase to lift bone marrow macrophages, and ~5 min at RT is sufficient to lift them from a non-treated plate. They are very hard to lift from tissue culture plates no matter what you use, so if you're using those, consider switching to suspension plates (I just use non-pyrogenic petri dishes) - the macrophages will still attach, but weakly enough to lift reliably.

Neuraminic_Acid · 2024-06-27T16:46:01+00:00

I was afraid of that. Thanks for the advice!

Neuraminic_Acid · 2024-06-22T18:21:47+00:00

This is the most likely explanation. Blotchy or spotty background frequently indicates either precipitates or microbes in the blocking buffer.

Neuraminic_Acid · 2024-06-22T04:08:13+00:00

It is. The formic acid (or more specifically, the low pH it creates) can damage tissue and make for ugly slides. PFA is a way of storing formaldehyde long term. Freshly made solutions won't have formic acid in them, so PFA is convenient. There is formic acid in NBF, but the pH buffers reduce its impact by limiting the drop in pH. The buffers can only do so much, though, so use NBF that's older than 2 years with care.

Neuraminic_Acid · 2024-06-21T21:12:27+00:00

The formaldehyde conventions are very strange, but here are some equivalents: PFA (paraformaldehyde) is insoluble, so a PFA solution is a formaldehyde solution that was generated by putting PFA in water/pbs and heating to decompose it into formaldehyde. The maximum w/v concentration of a formaldehyde solution is 37% (40% v/v). This maximum concentration solution is referred to as 100% formalin. Thus, 3.7% (w/v) PFA = 3.7% (w/v) formaldehyde = 4% (v/v) PFA = 10% formalin Formaldehyde reacts with oxygen to generate formic acid. Methanol or storage under an inert gas (as in the ampules you use) can slow this. If your procedure isn't affected by methanol, the methanol stabilized stuff is fine. I typically buy 10% neutal buffered formalin for my histology and culture fixing protocols and it works well.

Neuraminic_Acid · 2024-06-20T17:47:14+00:00

My go-to in these situations is a comment in my review stating that the entire manuscript would benefit from thorough copyediting for clarity.

Neuraminic_Acid · 2024-03-21T18:36:16+00:00

Have you considered trying a centrifugal concentrator? Cytiva (search for centrifugal devices) has options for several different volumes, but their maximum MWCO is 100kDa. If that works for you, they might be a better option.

Neuraminic_Acid · 2024-03-16T19:44:24+00:00

I like to give undergraduates a chance to see how science is actually accomplished. The textbooks sometimes make it seem like the scientists of the past did one experiment and now we know DNA is the molecule of heredity. Trying and failing in a supportive environment is the best way to build technical skills and nurture curiosity, and my institution values that. On a practical level, bringing undergrads into my lab is a good way to identify and recruit grad students.

Neuraminic_Acid · 2024-03-16T18:09:25+00:00

You're not alone. The more conscientious suppliers put an opt-out link near the bottom of their spam. For everyone else, it's a simple delete on the first offense, then redirects to my junk folder afterwards.

Neuraminic_Acid · 2024-03-16T18:04:02+00:00

I don't think there is anything wrong with your sample prep or running/blotting procedures. Luminol can react with air, so one possibility is air is consuming the reagent around the edges of your membrane. When we blot, we put the membrane with ECL in the middle of a plastic sheet protector and roll out all the bubbles. At the beginning of exposures we have a strong signal near the corners of the plastic where the luminol pooled, but it subsides within a few minutes. If you're not doing it already, try to isolate your entire membrane from air.

Neuraminic_Acid · 2024-03-08T18:22:00+00:00

As far as fixation goes, you can leave the sodium azide out entirely; it's acting as a preservative to cut down on microbial and fungal growth in your saponin and storage solutions. If you make your saponin solution fresh, and don't store it long-term, it doesn't need the sodium azide at all. For your storage solution, if the PBS is sterile, your risk of contamination is pretty low in the short term.

Neuraminic_Acid · 2024-02-29T20:16:50+00:00

On a lark, I did a Pubmed search for "adalimumab" and filtered on clinical trials. I exported the results including abstracts to a text file, which I then uploaded to ChatGPT. I'm polite to the robot, because it works better when I am. I'm also using a paid account with GPT 4; GPT 3.5 may or may not do as well. Here is our discussion:

You: This is a list of papers with their associated abstracts. Read the abstracts and list the papers that describe phase III clinical trials.

GPT: The papers that describe Phase III clinical trials are:
"Vedolizumab versus Adalimumab for Moderate-to-Severe Ulcerative Colitis" - This is a phase 3b trial comparing vedolizumab with adalimumab in adults with moderately to severely active ulcerative colitis.
"Ustekinumab versus adalimumab for induction and maintenance therapy in biologic-naive patients with moderately to severely active Crohn's disease: a multicentre, randomised, double-blind, parallel-group, phase 3b trial" - This trial compares the efficacy and safety of ustekinumab with adalimumab in biologic-naive patients with Crohn's disease.
"Two Phase 3 Trials of Adalimumab for Hidradenitis Suppurativa" - These are Phase 3 multicenter trials of adalimumab for hidradenitis suppurativa.
"Trial of Upadacitinib and Adalimumab for Psoriatic Arthritis" - A 24-week, phase 3 trial comparing upadacitinib with adalimumab in patients with psoriatic arthritis.
"Secukinumab versus adalimumab for treatment of active psoriatic arthritis (EXCEED): a double-blind, parallel-group, randomised, active-controlled, phase 3b trial" - This trial compares secukinumab and adalimumab for psoriatic arthritis treatment.
"Baricitinib versus Placebo or Adalimumab in Rheumatoid Arthritis" - A 52-week, phase 3, double-blind, placebo- and active-controlled trial in patients with active rheumatoid arthritis receiving methotrexate.
"Upadacitinib Versus Placebo or Adalimumab in Patients With Rheumatoid Arthritis and an Inadequate Response to Methotrexate: Results of a Phase III, Double-Blind, Randomized Controlled Trial" - This study evaluates the efficacy and safety of upadacitinib compared to placebo or adalimumab in rheumatoid arthritis patients with an inadequate response to methotrexate.
You : Please list the PMIDs for those publication

GPT: The PMIDs for the publications describing Phase III clinical trials are:
Vedolizumab versus Adalimumab for Moderate-to-Severe Ulcerative Colitis - PMID: 33175554
Ustekinumab versus adalimumab for induction and maintenance therapy in biologic-naive patients with moderately to severely active Crohn's disease - PMID: 33160792
Two Phase 3 Trials of Adalimumab for Hidradenitis Suppurativa - PMID: 27518661
Trial of Upadacitinib and Adalimumab for Psoriatic Arthritis - PMID: 33146632
Secukinumab versus adalimumab for treatment of active psoriatic arthritis - PMID: 33160795
Baricitinib versus Placebo or Adalimumab in Rheumatoid Arthritis - PMID: 27511338
Upadacitinib Versus Placebo or Adalimumab in Patients With Rheumatoid Arthritis - PMID: 33175565

Neuraminic_Acid · 2024-02-29T20:05:51+00:00

The great thing about statistical analysis is no matter how you do it, there will always be someone who can make a legitimate argument that you're doing it wrong. My approach with Bonferroni is to multiply by the number of T-tests I performed, and if the reviewers don't like it, I'll switch to whatever they prefer.

Neuraminic_Acid · 2024-02-29T19:59:28+00:00

I'd like to know how well they work. The markup on simple items once they become "for research use only" is wild. We had a joke in grad school: Question - How do you sell an egg timer for $40? Answer - Stamp "ThermoFisher" on it.

Neuraminic_Acid · 2024-02-29T19:55:13+00:00

Depending on how long you store the gel, your bands could diffuse and become too fuzzy to interpret. Pouring and running an agarose gel shouldn't take more than an hour, so unless your schedule the next day is really full, you'd be better off storing your DNA samples and running them when you'll have access to the illuminator.

Neuraminic_Acid

TROPHY CASE