Hello! Do you know any method for tRNA isolation using E.coli? I know that there are a lot of commercial kits but I prefer do it using reagents in lab. What is your experience?
Ping Wu
Cell Lysis: Use a suitable lysis buffer (e.g., Trizol or a similar reagent) to break open the bacterial cells and release the RNA. Ensure the buffer contains guanidine isothiocyanate or similar agents to denature RNases, which could degrade RNA during the extraction. RNA Extraction: After lysis, separate RNA from other cellular components by phenol-chloroform extraction. The aqueous phase will contain RNA, including tRNA. RNA Precipitation: Precipitate the RNA by adding isopropanol or ethanol and incubate at -20°C to enhance precipitation. Purification: To specifically enrich tRNA, use gel-based methods like size-exclusion chromatography, or affinity-based techniques targeting tRNA’s unique features, such as its 3' CCA tail.
Certainly! To isolate tRNA from E. coli, you can follow a multi-step process involving cell lysis, RNA extraction, and subsequent purification. Here's a general approach:
- Alternatively, if you’re looking to avoid using commercial kits, the above approach can be modified with reagents available in your lab. A crucial step is to avoid RNase contamination to ensure tRNA integrity. For more streamlined processes, you may also want to consider outsourcing to CRO companies like ThermoFisher, BOC Sciences, MedChemExpress, which offer professional RNA extraction services.
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