I am having a lot of trouble with in vitro transcription and wonder if
anyone can help. The reaction is to produce Dig-labelled riboprobes.
I am using all Roche reagents except for RNAguard. I follow the
protocol given with the SP6 and T7 transcriptases (Roche). My first
problem seems to be with the DNaseI which despite increasing
incubation time still seems not to be removing all the template DNA.
Following this, the clean up kits I have been using seem to be very
ineffective. I initially used Qiagen RNeasy kit cleanup protocol but
this has a cut off of 200 bases and some of my transcripts are
borderline for this. I couldn't get much, if any, product after the
kit so I then got the Qiagen RNA/DNA kit which processes transcripts
down to about 25 bases. This kit, however, has many more steps
including a reprecipitation which seems to be giving an infinitely low
My most recent reaction was for 4 transcripts (2 genes, sense and
antisense) and I got a different result for each. One appeared not to
have transcribed, the next did but had no product after cleanup
(RNA/DNA kit). A third had 4 bands on the gel following cleanup and
the last potentially produced a usable probe which I am currently
testing. I carried out the same procedure on each, at the same time.
I know I included all the correct reagents etc and am also careful a
out RNase contamination.
I am qutie new to molecular biology (marine biologist by trade) Is
this just a norotiously difficult reaction or am I missing something
obvious? Any suggestions?!