I have been doing Northern blots for three years now and in the last two months I have been unable to purify my RNA probe. I transcribe the RNA with a T7 polymerase. (I know transcription works because I ran the hot probe on a gel and saw a band.) For cleanup in the past I have always used the GE Microspin G-50 column. I recently purchased a new kit and I have not been able to recover much RNA. I ran a 50uL volume reaction (20uL reaction + 30uL water) and the 20uL reaction through the columns. 

I ran the 20uL reaction through the column and collected the eluate. I then added another 20uL to the G50 column and spun it again. I collected a total of 5 washes from the column using my previously described method. I also ran a separate transcription reaction through an RNA clean up kit that works on RNA adhesion to the column instead of separation by weight. I washed the kit column twice.

To check the amount of RNA for every wash of each column I ran the RNA on a gel and then exposed it.

Results: The in vitro transcribed RNA (before clean up) had a strong signal. The washes for the columns had a weak signal in every lane. The column washes were ~100X weaker than the transcribed RNA.  Only the first wash for the kit column produced a distinct band.

Long story short, has anyone had these same problems with the G-50 columns for RNA isolation? If you do not use a G-50 column to clean up in vitro transcribed RNA, do you use phenol-choloroform?

I appreciate your input!!