In vitro transcription problems

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
yield.

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?!

Pamela

>

I'm not sure what your downstream application is, but I don't think
you need to do such an extensive cleanup. Certainly not for
hybridizing a blot. In the past, I've simply used a gel filtration
spin column (like the kind used for cleaning up sequencing reactions,
e.g. AutoSeq G-50 from Pharmacia) to clean up in vitro transcription
reactions. I never noticed problems with yield loss with these.

Mike
--

Michael L. Sullivan, Ph.D
Research Plant Molecular
Geneticist
U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5397 Phone
(608) 264-5147 Fax
---

In article <[Only registered users see links. ]>, AudioHELP
<[Only registered users see links. ].uk> wrote:


Were any of the four bands the expected size for the full length
transcript? Many templates are prone to producing prematurely
terminated products, does this account for the additional bands?

I also agree with the other poster that a simple size exclusion spin
column is a reasonable approach. I'm not familiar with the kits that
you mention, but Dig-labelled probes don't behave exactly the same way
as unmodified RNAs. However, I'm puzzled by the failure of the DNaseI
treatment, as this enzyme step is usually pretty effective.

Good luck,

Pam