In my lab, i am doing the in vitro transcription experiments to get our tRNA, i do the PCI-CI step, then refolding, but after refolding, i add the MgCl2 it becomes so cloudy. It's unlikely because of the MgCl2, since my other tRNA use the same reagent still transparent.
Cloudiness after MgCl₂ addition suggests that your specific tRNA may be misfolding or aggregating upon refolding. This could be due to incomplete folding before Mg²⁺ is added, residual PCI contaminants, or the intrinsic instability of the transcript. Try purifying the tRNA via PAGE, adjusting the folding protocol (e.g., slower cooling), and titrating down the MgCl₂ concentration to minimize aggregation.
There is a substantial release of pyrophosphate upon T7 transcription, and it precipitates easily in the presence of excessive Mg(2+). Pyrophosphate is not removed by phenol-chloroform extraction or ethanol precipitation, thus it will remain in your RNA prep until the refolding step. It may happen that your other tRNA transcription reaction is not that efficient, and the final concentration of pyrophosphate is not as high as in the reaction mixture which eventually gets cloudy. Also, if you use GMP as a booster to increase transcription rate and yield, it may also precipitate at elevated Mg(2+) concentrations. The easiest way to get rid of pyrophosphate (and GMP if present) is to perform buffer exchange right after the transcription reaction stop. You can do it with centrifugal membrane filter units with MWCO around 10 kDa, or use small gel-filtration column with Sephadex G-25 or similar matrix.
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