RNA extration from plant - Lower yield... (Jul/30/2009 )
I am extracting total RNA from mango fruits using the extraction buffer having the composition of 100mM Tris-Cl (pH 8.2), 1.5M NaCl, 30mM EDTA (pH 8), 2% CTAB. Previously (before 2 yrs) according to the same standardized protocol, we used to get very good yield (ca 400 ug per gram of tissue) but now I am getting yield like 3 ug/g tissue. I went back and checked the pH of stock solutions and found pH 8.1 (instead of 8.2) for tris, 7.6 (instead of 8) for EDTA and 5 (instead of 5.4) for sodium acetate (used during final precipitation along with isopropanol). The first precipitation is with 3M final concentration of lithium chloride. Can the lowered yield be because of different pH than desired?
The pH is quite important, if you have the wrong pH you can skew your extraction towards DNA rather than RNA.