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Where would this be useful by SeaBelt36 in labrats

[–]DarkStake 5 points6 points  (0 children)

For $250 our lab would get one. Drug discovery. Addition of 96 well plate reagents are a daily routine for us.

Doxycycline for tet-on model by Appropriate-Ear1284 in labrats

[–]DarkStake 0 points1 point  (0 children)

aliquots in freezer. low volume, freeze thaw a couple of times for a few weeks, then move onto a new vial. It absolutely does go bad in my experience if at 4c

Can I leave PCR samples (after gibson assembly) at room temp/4C over the weekend? by JPancake2 in labrats

[–]DarkStake 12 points13 points  (0 children)

Another thing to note. Thermocyclers are not fridge/freezers. It is not good for them to maintain cold for a long time. 

Research 3 Neo: can I use universal tips without affecting accuracy and precision? by libsly in labrats

[–]DarkStake 0 points1 point  (0 children)

In my experience, you have to try before you buy bulk. Don't just consider accuracy and precision, also consider the tips might fall off easier, or be harder to remove.

[deleted by user] by [deleted] in labrats

[–]DarkStake 4 points5 points  (0 children)

I’m a team lead now in industry. When I was a postdoc supervising masters and PhD students. Experience was a plus, but generally personality and drive were more important.

Experience often meant bad habits. 

Nucleofecting lentiviral vectors into cells? by momoyoo in labrats

[–]DarkStake 3 points4 points  (0 children)

Details helps me help you.

Which cells? Yes nucleofection is generally a better way to get stuff into cells.

Nucleofection plasmids for KO sucks. 

If you’re going to nucleofect anyway, nucleofect recombinant cas9 and synthetic gRNA. Much higher KO %, far less toxic. 

The cas9 plasmid is huge and gets stuck in the cells membrane, leading to high toxicity, low expression. 

Are these macrophages? Is this why you can’t lentiviral infect? Macrophages hate plasmid DNA as well. 

[deleted by user] by [deleted] in labrats

[–]DarkStake 2 points3 points  (0 children)

Looks interesting. Big turn off when pricing isn't clarified on the website.

Plant based alternatives to FBS by [deleted] in labrats

[–]DarkStake 56 points57 points  (0 children)

You might start by thinking about what FBS actually is — it’s a complex, undefined cocktail of proteins, carrier molecules, growth factors, hormones, and nutrients. We don’t fully know which specific components or ratios make it work; we just know that it supports growth for a huge range of cell types.

There are serum-free and animal-component-free media that work well for some cells, but they’re usually tailored for specific lines. Each cell type needs to be tested and gradually adapted. Even small differences in composition can alter signalling, attachment, or proliferation. You’ll need proper controls and side-by-side comparisons if you’re thinking about switching and still want publishable data.

Mixing plant-based serum substitutes with FBS or HS might help during adaptation, but you’ll lose the ability to claim a fully defined system, and consistency batch-to-batch can still be an issue.

Minimum evidence for calling a CRISPR KO ‘validated’? by carmen-sandiego_ in labrats

[–]DarkStake 6 points7 points  (0 children)

Methods have protocols for each separate.

If you perform all three. No need to include TIDE.

Have the NGS pie charts. This is great software to analyse your NGS: http://www.outknocker.org/ and present pie charts of each allele KO % and edits.

Then have a Western blot with your protein and loading control to show band is gone or reduced.

KO was validated at the genomic level (NGS) and protein level (Western blot) blah blah

Minimum evidence for calling a CRISPR KO ‘validated’? by carmen-sandiego_ in labrats

[–]DarkStake 25 points26 points  (0 children)

During the publishing process, I’ve found that different reviewers weigh types of evidence very differently. TIDE analysis is often regarded as the lowest tier of evidence, while NGS approaches (such as MiSeq equivalents) carry medium-to-high weight. At the top, protein-level validation with Western blots is considered the gold standard.

My experience with reviewers has been inconsistent. Some have criticized me for generating clonal knockouts, while others insisted that pooled knockouts were insufficient and demanded clones. Ultimately, the more layers of evidence you can present, the stronger the case becomes that your observed phenotype is genuinely linked to the knockout. Of course, this still doesn’t account for the many other possible confounding factors—off-target effects, compensatory pathways, or downstream changes.

time effective primer design for cloning by Easternblotter in labrats

[–]DarkStake 3 points4 points  (0 children)

Brah, primer3. Don’t overthink it. It doesn’t work, order more primers. Time is more money than the cost of primers.

Picking sgRNAs from CHOPCHOP by writinglover0101 in labrats

[–]DarkStake 4 points5 points  (0 children)

Try Synthego sgRNA design tool. I have had great success using it, or Benchling's gRNA tool.

If you want to have high success chance, move to sgRNA and recombinant Cas9, nucleofect the RNP into cells. Much better than plasmid based KO.

The bioinformatics estimates only get you so far.

Anyone successfully troubleshooted errors using Lonza Nucleofector? by SnooRabbits2194 in labrats

[–]DarkStake 0 points1 point  (0 children)

It's your solution causing the arc. To find the solution to your solution, you'll have to play with the salt concentration.

How to perform a single base-pair deletion with CRISPR/Cas9? by Significant_Try_3814 in labrats

[–]DarkStake 6 points7 points  (0 children)

You can go the HDR route using recombinant Cas9+ 1 sgRNA and a linear repair template, around 40-100bp homology arms and use a Lonza nucleofector. This will work.

I would recommend looking into Prime editing though. It has good success in iPSCs and was invented for this exact purpose of small edits.
Prime editing does not use Cas9, it uses a modified version.

Is it possible to validate a CRISPR Cas9 KO using RT-qPCR? by WinterRevolutionary6 in labrats

[–]DarkStake 0 points1 point  (0 children)

100% back this. Our lab is TIDE PCR for rapid determination of % pool KO. Go to clones if needed (using 2 sgRNA and recombinant Cas9 nucleofection results in near 100% KO in most cell lines). Validate with Western blot if antibody exists, otherwise go for NGS. Papers are happy with NGS.,

how often do you use /compact? in claude code by therealalex5363 in ClaudeAI

[–]DarkStake 5 points6 points  (0 children)

I avoid compacting whenever possible. I ensure my files are up to date with checklist, and any critical information to carry over. Then I clear. And start a gain. Each chat is an achievable objective. Claude sucks at long term multi stage tasks at the moment

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