I am using polysome sucrose gradient coupled with QPCR in order to investigate the transcription and translation regulation of certain genes under various conditions. *I know I could do ribo-seq but its too expensive for my lab*
However, when extraction the mRNA (polysome) from the sucrose gradient fractions, my 260/230 number is never past 1.5 and much of the time is near 1.1. My strategy to put it briefly: 300 mg fresh tissue ==> polysome extraction ==> sucrose gradient ==> fractionation of gradient ==> isopropanol precipitation with glycoBlue at -80˚C 30 minutes ==> DNAse treatment ==> phenol:chloroform:isoamy alcohol pH 4.8 (25:24:1) extraction, ==> by ethanol precipitation -20˚C O.N> ==> 70% ethanol washes x2.
Is this the best method to use? Should I expect higher 260/230 read outs?
Thanks to anyone with insight into this problem.
- Badges
- Science method