Due to the excessive number of times that these samples have been DNased, I’m concerned that the buffer is becoming too concentrated. So, I’m performing an EtOH precip on them to clean them up and then proceed with another round of DNase treatment.
0.1 volumes of 3M sodium acetate (pH = 5.2) were added to each sample. Then, 2 volumes of 100%, ice-cold EtOH were added. Samples were mixed and stored @ -20C for 2hrs. Samples were then centrifuged @ 4C, 16,000g for 30mins. Supe removed. Added 1mL of 70% EtOH and then centrifuged samples @ 4C, 16,000g for 15mins. Supe was removed, tubes were spun briefly to pool residual EtOH and the remaining EtOH was removed. RNA was resuspended in 20uL of 0.1% DEPC-H2O and spec’d.
Results: The following samples appear to have no RNA after precipitation:
06:5-34, 37; 06:6-44, 45, 46, 47, 52; 08:4-9, 10, 13, 15-18; All of the 07:12 samples.
Since I did not DNase the 07:12 samples, I can’t say whether or not this result was expected (e.g. due to low initial concentration). The others that have no remaining RNA are a surprise and is a bit disconcerting.
Will take fresh RNA aliquots of those samples for DNasing. For those samples still having RNA post-recip, I will just use them as they are, as most concentrations are in the recommended range for DNasing, according to the Ambion Tubro DNA-free kit.