u/DiosAnthos

Hi everyone,

I’m currently working with recombinant proteins expressed in E. coli with fusion tags (mainly His-tag and thioredoxin/TRX), and I’m trying to decide on the most reliable protease for tag removal.

I’d be curious to hear your experience with different options, especially in terms of:

• cleavage efficiency
• specificity (off-target cleavage issues)
• performance under different buffer conditions
• overall ease of use in routine purification workflows

From what I’ve seen, TEV protease and HRV 3C (PreScission) seem to be the most commonly used, but I’d love to hear real-world opinions on what actually works best in practice.

Also, has anyone here tried using in-house produced / self-purified proteases (e.g., expressing and purifying TEV or other proteases themselves)? If so, would you be willing to share your general experience or a high-level overview of your purification strategy?

Any tips, comparisons, or pitfalls to avoid would be greatly appreciated.

Thanks in advance!

Hello everyone,
Has anyone here performed DNA extraction in a 96-well plate format (high-throughput) from plant material (e.g. Arabidopsis or other plants)?
I am trying to adapt the classic Edwards method to a 96-well format and I am wondering what the best approach is for the tissue disruption / homogenization step in the plate.
I am especially interested in:
how you physically grind or disrupt leaf tissue in the wells (leaf punch, pipette tip, pestle, bead beating, etc.)
whether this is done before or after adding extraction buffer
whether multichannel pipettes are used for grinding or if separate tools are preferred
what setups work best for larger sample numbers (e.g. 500–1000 samples)
I would like to avoid cross-contamination issues while maintaining reasonable throughput.
Any protocols, tips, or even photos of your setup would be highly appreciated.
Thank you!

Hey!

I'm trying to generate mutants of three isoforms of a gene that differ in their TSS (transcription start site) using the CRISPR-Cas method.

I'm designing everything according to the protocol at this link: https://bio-protocol.org/en/bpdetail?id=3796&type=0

I then transformed the WT plants using the floral dip method under vacuum.

I selected the seeds of T1 generation that glow red under a fluorescence microscope. However, after sowing and genotyping the plants, it turns out that none of the plants are mutated. Do you have any ideas on how to solve my problem? Design different guides? Switch to a different system? I would appreciate any advice or ideas

I was thinking about prolong activity of Cas9 by leaving glowing seeds by one generation, but I am risking higher off target cutting. What do you think?