How to remove pigment from your extracted DNA of peanut leaves?

I am analyzing WGS of some bacterial strains some of them have been reported to have plasmids, I extracted the DNA using bashing beads and spin column. I then sent those samples to sequencing and i obtained the data. Is it posible that I have sent to sequencing my gDNA and plasmids in case the bacteria had them?

Hi, i was wondering if anyone could help me or share any methods/protocol on how to prepare hoagland solution as a source of fertilizer?

Hi all,

We are currently working on extracting microbial DNA from clinical blood samples (from BacT/ALERT bottles) for downstream NGS applications. We’re using a commercial kit for microbial DNA extraction but are encountering issues with obtaining pure DNA. Despite performing additional purification steps, we are unable to achieve the recommended 260/230 ratio within the desired range.

We’ve tried multiple kits, including the Macherey-Nagel NucleoSpin kit, the Invitek DNA extraction kit, and the MP Biomedicals kit, but we still can't obtain the desired purity.

Could anyone suggest ways to obtain purer microbial DNA or provide insights on whether the blood samples from BacT/ALERT bottles might be causing any issues? Any advice or suggestions would be greatly appreciated.

Thank you in advance, Sumana

I’m working with a recombinant P450 enzyme that I’ve directed to the periplasmic space using a signal peptide. Since I will use this enzyme for downstream applications, maintaining its activity is critical.

Unfortunately, osmotic shock has given me very poor yields — only trace amounts of protein. In contrast, sonication significantly improves the yield, but a large portion of the protein still remains in the pellet.

I’m using a pulse sonicator (adjustable duty cycle), and I plan to optimize extraction using various amplitudes, pulse durations, and total times. However, I have a few questions before setting up my trials:

1. Does sonication damage enzymes like P450s? Have you had experiences where sonication compromised enzymatic activity?

2.Should I suspend my pellet in TES buffer or binding buffer before sonication? TES seems to help stabilize the periplasmic environment, but I’m not sure if it provides a real advantage.

3.For pulse settings: – Would you recommend a 0.2 duty cycle (e.g., sonicate 20% of each second, 80% rest), and optimize amplitude and time? – Or should I go with a 1.0 duty cycle (continuous sonication) but in short bursts like 10 sec on / 30 sec off?

Any experience or suggestions regarding buffer choice and sonication parameters for periplasmic protein extraction would be greatly appreciated.

Does anyone have experience using PEI to precipitate DNA from bacterial cell lysate during protein purification while working in buffers containing 6M guanidine hydrochloride? My lysate must be in such buffer, otherwise my intrisically disordered protein gets cut during the whole process by proteases, but I also need something that will help me get rid of the DNA and I was recommanded PEI.

Dear colleagues,

I need key information regarding the structural stability of secondary metabolites. If you have any details on the stability of secondary metabolites such as phenolic compounds, alkaloids, and terpenes, I would greatly appreciate it if you could share them with me. This will help me conduct a comprehensive comparison of these metabolites in terms of stability.

Thank you very much for your time and consideration.

I'm working on a protein with a GST tag followed by a His tag. To remove the GST tag, I performed thrombin digestion at 4°C in the buffer containing 500 mM NaCl, 50 mM Tris (pH 8), and 5 mM β-mercaptoethanol. At the 12-hour mark, cleavage was only partial, so I extended the duration of the digestion to 4 more hours (16 hours in total). Further, I performed NiNTA Re-affinity. The GST was eluted during elution, and my protein was detected in the flow-through and column wash. However, the total yield of the protein was very low.

Previously, in the buffer with only 20 mM Tris and 5 mM β-mercaptoethanol (no NaCl), complete digestion occurred at the 12th hour.

Could the high salt concentration (500 mM NaCl) have affected the thrombin activity? What other factors might have affected the cleavage?

Dear colleagues, if you have any useful information on this topic, please share it with me. Thank you for your support.

I have to do various types of phenotypic and physiological assays after the abiotic stress treatment on the transgenic tobacco plants. For that i have to grow them in plant media containing the stress factors like mannitol, nacl etc. Should the media also contain sucrose?