Formaldehyde Gel Electrophoresis on non-denatured/denatured RNA - No/Faint RNA bands after denatration (Sep/06/2007 )

Hi,

I have some problems with RNA electrophoresis.

Below is what I had done.

Solutions for the gel electrophoresis was prepared the day before the run.

Electrophoresis tanks, gel trays and glassware were soaked in 0.5% SDS overnight and rinsed thoroughly with 0.1% DEPC.

1.2% FA gel was prepared for the run ( protocol adapted from RNeasy Mini Handbook) . Prior to running , FA gel is equilibrated in 1x FA gel running buffer.

Duplicates of 2 to 5ug of RNA template were prepared, one denatured , one non-denatured.


Attached are the gel pic and RNA ladder. The 1st 6 lanes including the RNA ladder [after mixing with 5x loading buffer (eg: 8ul of sample + 2ul of buffer)] are heat -denatured for 1min at 65C, cooled for 5min. The tubes were spinned before loading into the wells.

I tried 1min heat denaturation as at 3min, there's no visible bands at all

The last 6 lanes together with the RNA ladder are non-denatured.

Someone pls help ...

The rna looks quite degraded. Try to minimize degradation by the usual means (improve rna preps especially avoiding protein contamination, clean well all instruments, use depc-treated water and buffers, bake overnight at 180 °C glassware, etc).